U.S. patent application number 10/371067 was filed with the patent office on 2004-01-29 for nanostructures containing antibody assembly units.
Invention is credited to Goldberg, Edward B., Makowski, Lee, Williams, Mark K..
Application Number | 20040018587 10/371067 |
Document ID | / |
Family ID | 30773735 |
Filed Date | 2004-01-29 |
United States Patent
Application |
20040018587 |
Kind Code |
A1 |
Makowski, Lee ; et
al. |
January 29, 2004 |
Nanostructures containing antibody assembly units
Abstract
Nanostructures are made that include at least one species of
assembly unit that is an antibody assembly unit in which at least
one joining element, structural element or functional element is an
antibody or antibody fragment. Antibody assembly units may have
more than one antibody element. In addition, the antibody assembly
units may contain non-antibody structural, functional or joining
elements. The nanostructure is suitably prepared using a staged
assembly method. In this method, a nanostructure intermediate
having at least one unbound joining element is contacted with an
assembly unit having a plurality of different joining elements.
None of the joining elements of the assembly unit can interact with
itself or with another joining element of the same assembly unit.
However, one of the joining elements of the assembly unit can
interact with the unbound joining element of the nanostructure
intermediate, so that the assembly unit is non-covalently bound to
the nanostructure intermediate to form a new nanostructure
intermediate for use in subsequent cycles. Unbound assembly units
are removed and the cycles is repeated for a sufficient number of
cycles to form a nanostructure. The assembly units in at least one
cycle are antibody assembly units.
Inventors: |
Makowski, Lee; (Hinsdale,
IL) ; Williams, Mark K.; (Revere, MA) ;
Goldberg, Edward B.; (Newton, MA) |
Correspondence
Address: |
OPPEDAHL AND LARSON LLP
P O BOX 5068
DILLON
CO
80435-5068
US
|
Family ID: |
30773735 |
Appl. No.: |
10/371067 |
Filed: |
February 21, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10371067 |
Feb 21, 2003 |
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10080608 |
Feb 21, 2002 |
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10371067 |
Feb 21, 2003 |
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10136225 |
Apr 29, 2002 |
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10136225 |
Apr 29, 2002 |
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09236949 |
Jan 25, 1999 |
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6437112 |
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09236949 |
Jan 25, 1999 |
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08542003 |
Oct 12, 1995 |
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5864013 |
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08542003 |
Oct 12, 1995 |
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08322760 |
Oct 13, 1994 |
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5877279 |
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Current U.S.
Class: |
435/68.1 ;
530/387.1 |
Current CPC
Class: |
B82Y 5/00 20130101; B82Y
10/00 20130101 |
Class at
Publication: |
435/68.1 ;
530/387.1 |
International
Class: |
C12P 021/06; C07K
016/00 |
Claims
What is claimed is:
1. A method for staged assembly of a nanostructure comprising: (a)
contacting a nanostructure intermediate comprising at least one
unbound joining element with an assembly unit comprising a
plurality of different joining elements, wherein: (i) none of the
joining elements of said plurality of different joining elements
can interact with itself or with another joining element of said
plurality, and (ii) a single joining element of said plurality and
a single unbound joining element of the nanostructure intermediate
are complementary joining element, whereby the assembly unit is
non-covalently bound to the nanostructure intermediate to form a
new nanostructure intermediate for use in subsequent cycles; (b)
removing unbound assembly units; and (c) repeating steps (a) and
(b) for a sufficient number of cycles to form a nanostructure,
wherein the assembly unit in at least one cycle comprises an
antibody or antibody fragment or a binding derivative thereof.
2. The method of claim 1, wherein the nanostructure intermediate
comprises a surface-bound initiator assembly unit.
3. The method of claim 1, comprising the additional step of capping
the nanostructure with at least one capping unit.
4. The method of claim 1, wherein the antibody assembly unit
comprises a structural element and a first joining element
comprising an antibody or antibody fragment or a binding derivative
thereof.
5. The method of claim 4, wherein the structural element is
covalently linked to the first joining element and to a second
joining element.
6. The method of claim 5, wherein the second joining element
comprises an antibody or antibody fragment or a binding derivative
thereof.
7. The method of claim 4, wherein the antibody assembly unit
further comprises a functional element.
8. The method of claim 7, wherein the functional element comprises
a photoactive molecule, photonic nanoparticle, inorganic ion,
inorganic nanoparticle, magnetic ion, magnetic nanoparticle,
electronic nanoparticle, metallic nanoparticle, metal oxide
nanoparticle, gold nanoparticle, gold-coated nanoparticle, carbon
nanotube, nanocrystal, nanowire, quantum dot, peptide, protein,
protein domain, enzyme, hapten, antigen, biotin, digoxygenin,
lectin, toxin, radioactive label, fluorophore, chromophore, or
chemiluminescent molecule.
9. The method of claim 7, wherein the functional element comprises
an antibody or antibody fragment or a binding derivative
thereof.
10. The method of claim 1, wherein the antibody assembly unit
comprises a functional element and a joining element comprising an
antibody or antibody fragment or a binding derivative thereof.
11. The method of claim 10, wherein the functional element
comprises a photoactive molecule, photonic nanoparticle, inorganic
ion, inorganic nanoparticle, magnetic ion, magnetic nanoparticle,
electronic nanoparticle, metallic nanoparticle, metal oxide
nanoparticle, gold nanoparticle, gold-coated nanoparticle, carbon
nanotube, nanocrystal, nanowire, quantum dot, peptide, protein,
protein domain, enzyme, hapten, antigen, biotin, digoxygenin,
lectin, toxin, radioactive label, fluorophore, chromophore, or
chemiluminescent molecule.
12. The method of claim 10, wherein the functional element
comprises an antibody or antibody fragment or a binding derivative
thereof.
13. The method of claim 1, further comprising the step of
post-assembly conversion of specific non-covalent interactions of
complementary joining elements to covalent linkages. whereby the
linkages are stabilized.
14. The method of claim 1, wherein the antibody assembly unit
comprises an antibody, antibody binding derivative or antibody
binding fragment selected from the group consisting of IgG, IgM,
IgE, IgA, and IgD and derivatives and fragments thereof.
15. The method of claim 1, wherein the antibody assembly unit
comprises a chimeric antibody, antibody binding derivative or
antibody binding fragment.
16. The method of claim 1, wherein the antibody assembly unit
comprises a multispecific antibody, antibody binding derivative or
antibody binding fragment.
17. The method of claim 1, wherein the antibody assembly unit
comprises Fab or F(ab').sub.2 antibody fragments.
18. The method of claim 1, wherein the antibody assembly unit
comprises Fab or F(ab').sub.2 antibody fragments.
19. The method of claim 1, wherein a complementary joining pair is
formed from assembly units exhibiting an idiotope/anti-idiotope
interaction.
20. The method of claim 1, wherein a complementary joining pair is
formed from assembly units exhibiting an antigen/antibody
interaction.
21. A nanostructure formed from a plurality of species of assembly
units comprising a plurality of different joining elements forming
a plurality linkages between the assembly units, said assembly
units including a first assembly unit comprising an antibody or
antibody fragment, or a binding derivative thereof.
22. The nanostructure of claim 21, wherein the antibody or antibody
fragment, or a binding derivative thereof in the first assembly
unit is present as a joining element.
23. The nanostructure of claim 22, wherein the first assembly unit
further comprises a functional element.
24. The nanostructure of claim 23, wherein the functional element
comprises a photoactive molecule, photonic nanoparticle, inorganic
ion, inorganic nanoparticle, magnetic ion, magnetic nanoparticle,
electronic nanoparticle, metallic nanoparticle, metal oxide
nanoparticle, gold nanoparticle, gold-coated nanoparticle, carbon
nanotube, nanocrystal, nanowire, quantum dot, peptide, protein,
protein domain, enzyme, hapten, antigen, biotin, digoxygenin,
lectin, toxin, radioactive label, fluorophore, chromophore, or
chemiluminescent molecule.
25. The nanostructure of claim 24, wherein the functional element
comprises antibody or antibody fragment, or a binding derivative
thereof.
26. The nanostructure of claim 22, wherein the antibody or antibody
fragment, or a binding derivative thereof in the first assembly
unit is present as a functional element.
27. The nanostructure of claim 22, wherein the antibody or antibody
fragment, or a binding derivative thereof in the first assembly
unit is present as a structural element.
28. The nanostructure of claim 22, wherein the first assembly unit
comprises an antibody, antibody binding derivative or antibody
binding fragment selected from the group consisting of IgG, IgM,
IgE, IgA, and IgD and derivatives and fragments thereof.
29. The nanostructure of claim 22, wherein the first assembly unit
comprises a chimeric antibody, antibody binding derivative or
antibody binding fragment.
30. The nanostructure of claim 22, wherein the first assembly unit
comprises a multispecific antibody, antibody binding derivative or
antibody binding fragment.
31. The nanostructure of claim 22, wherein the first assembly unit
comprises Fab or F(ab').sub.2 antibody fragments.
32. The nanostructure of claim 22, wherein the first assembly unit
comprises Fab or F(ab').sub.2 antibody fragments.
33. The nanostructure of claim 22, wherein the nanostructure
comprises two assembly units linked by a complementary joining pair
formed from assembly units exhibiting an idiotope/anti-idiotope
interaction.
34. The nanostructure of claim 22, wherein the nanostructure
comprises two assembly units linked by a complementary joining pair
formed from assembly units exhibiting an antigen/antibody
interaction.
35. The nanostructure of claim 21, wherein the nanostructure is two
or three-dimensional.
Description
[0001] This application is a continuation-in-part of U.S. patent
application Ser. No. 10/080,608, filed Feb. 21, 2002, and of U.S.
patent application Ser. No. 10/136,225, filed Apr. 29, 2002, which
is a divisional of U.S. patent application Ser. No. 09/236,949,
filed Jan. 25, 1995, now U.S. Pat. No. 6,437,112, which is a
divisional of U.S. patent application Ser. No. 08/542,003, filed
Oct. 12, 1995, now U.S. Pat. No. 5,864,013, which is a
continuation-in-part of U.S. patent application Ser. No.
08/322,760, filed Oct. 31, 1994, now U.S. Pat. No. 5,877,279, all
of which are incorporated herein by reference.
TECHNICAL FIELD
[0002] The present invention relates to methods for the assembly of
nanostructures containing antibody or antibody fragment assembly
units for use in the construction of such nanostructures, and to
nanostructures containing antibody or antibody assembly units.
BACKGROUND OF THE INVENTION
[0003] Nanostructures are structures with individual components
having one or more characteristic dimensions in the nanometer range
(from about 1-100 nm). The advantages of assembling structures in
which components have physical dimensions in the nanometer range
have been discussed and speculated upon by scientists for over
forty years. The advantages of these structures were first pointed
out by Feynman (1959, There's Plenty of Room at the Bottom, An
Invitation to Enter a New Field of Physics (lecture), Dec. 29,
1959, American Physical Society, California Institute of
Technology, reprinted in Engineering and Science, February 1960,
California Institute of Technology, Pasadena, Calif.) and greatly
expanded on by Drexler (1986, Engines of Creation, Garden City,
N.Y.: Anchor Press/Doubleday). These scientists envisioned enormous
utility in the creation of architectures with very small
characteristic dimensions. The potential applications of
nanotechnology are pervasive and the expected impact on society is
huge (e.g., 2000, Nanotechnology Research Directions: IWGN Workshop
Report; Vision for Nanotechnology R & D in the Next Decade;
eds. M. C. Roco, R. S. Williams and P. Alivisatos, Kluwer Academic
Publishers). It is predicted that there will be a vast number of
potential applications for nanoscale devices and structures
including electronic and photonic components; medical sensors;
novel materials; biocompatible devices; nanoelectronics and
nanocircuits; and computer technology.
[0004] The physical and chemical attributes of a nanostructure
depend on the building blocks from which it is made. For example,
the size of these building blocks, and the angles at which they
join plays an important role in determining the properties of the
nanostructure, and the positions in which functional elements can
be placed. The art provides numerous examples of different types of
materials which can be used in nanostructures, including DNA (U.S.
Pat. Nos. 5,468,851, 5,948,897 and 6,072,044; WO 01/00876),
bacteriophage T even tail fibers (U.S. Pat. Nos. 5,864,013 and
5,877,279 and WO 00/77196), self-aligning peptides modeled on human
elastin and other fibrous proteins (U.S. Pat. No.5,969,106), and
artificial peptide recognition sequences (U.S. Pat. No. 5,712,366).
Nevertheless, there is a continuing need for additional types of
building blocks to provide the diversity which may be required to
meet all of the potential applications for nanostructures. The
present application provides a further class of building blocks
which can be used in homogeneous nanostructures containing building
blocks of only this class, or in heterogeneous nanostructures in
combination with building blocks of other classes.
SUMMARY OF THE INVENTION
[0005] The present invention provides nanostructures formed from a
plurality of species of assembly units. With some exceptions, such
as capping units, these assembly units comprise a plurality of
different joining elements. In the nanostructures of the invention,
the nanostructure includes at least one species of assembly unit in
which at least one joining, structural or functional element
comprises an antibody or antibody fragment. The antibody assembly
units may have one or more antibody or antibody fragment elements,
and in addition the antibody assembly units may contain other,
non-antibody, structural, functional and joining elements.
[0006] The nanostructure of the invention is suitably prepared
using a staged assembly method. In this method, a nanostructure
intermediate comprising at least one unbound joining element is
contacted with an assembly unit comprising a plurality of different
joining elements, wherein:
[0007] (i) none of the joining elements of said plurality of
different joining elements can interact with itself or with another
joining element of said plurality, and
[0008] (ii) a single joining element of said plurality and a single
unbound joining element of the nanostructure intermediate are
complementary joining elements.
[0009] As a result, the assembly unit is non-covalently bound to
the nanostructure intermediate to form a new nanostructure
intermediate for use in subsequent cycles. Unbound assembly units
are then removed and the process is repeated for a sufficient
number of cycles to form a nanostructure. In the method of the
invention, the assembly unit in at least one cycle comprises an
antibody assembly unit.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1. Diagram of an idiotope/anti-idiotope Fab-Fab
interaction. The diagram shows the .alpha.-carbon trace of two Fab
fragments interacting through idiotopic/anti-idiotopic interactions
(pdb entry 1CIC). The heavy lines represent the heavy chains and
the light lines represent the light chains of the Fab fragments.
Most of the idiotopic/anti-idiotopic protein binding interactions
occur between the loops of the heavy chains contained in the
complementarity determining region (CDR). In this case, the
association between Fabs results in a nearly linear
association.
[0011] FIG. 2. Line drawing representing the three-dimensional
structures of the .alpha.-carbon trace of a diabody (pdb entry
1LMK) (top) and a single chain Fv (scFv) antibody (pdb entry 2APA)
(bottom). For the monomeric scFv structure (bottom), heavy lines
represent the heavy chain and the light lines represent the light
chain. For the dimeric diabody structure (top), however, the heavy
lines represent both the heavy chain and light chain of one scFv,
while the light lines represent both the heavy and light chain of
the other scFv. scFv constructs that have the heavy-light variable
domains linked together by a longer peptide linkers form stable
monomers. Those with shorter linkers associate with a second scFv
molecule to form a bivalent diabody as shown. Note that the
immunoglobulin fold contained within both structures is very
similar. scFv and diabodies, or binding derivatives or binding
fragments thereof, can be used as the basic elements for the design
of assembly units.
[0012] FIG. 3. Diagram of single-chain Fv fragments (scFv). The top
half of the diagram shows monomeric, dimeric (diabody), trimeric
(triabody) and tetrameric (tetrabody) associations among
V.sub.H-linker-V.sub.L scFv fragments. The bottom half of the
diagram shows such associations among V.sub.L-linker-V.sub.H scFv
fragments. These associations between scFv domains are dependent
upon the length of the peptide linker joining the V.sub.H and
V.sub.L units. Longer peptide linkers (12-15 residues) favor
monomeric formation, whereas shorter linkers (0-5 residues), favor
multimeric structures. The linkage order of the V.sub.H and V.sub.L
genes also affects multimer formation, activity and stability of
the resultant scFv proteins. This type of recombinant antibody
represents one of the smallest functional antigen binding entities
derived from an IgG and can be utilized as the structural and
joining elements in assembly unit fabrication.
[0013] FIGS. 4(A-B). Diagram of two diabody units, Unit 1(A) and
Unit 2(B) and their associated genes. A. Unit 1 is an A.times.B
diabody in which the V.sub.H and V.sub.L domains of A define a
lysozyme isotopic antibody (D1.3) and in which the V.sub.H and
V.sub.L domains of B define a virus neutralizing idiotopic antibody
(730.1.4). In order to facilitate purification of the desired
diabody product, the gene encoding V.sub.HA and V.sub.LB includes a
hexahistidine tag, whereas the gene encoding V.sub.HB and V.sub.LA
does not. B. Unit 2 is B'.times.A' diabody in which the V.sub.H and
V.sub.L domains of B' define a virus neutralizing idiotopic
antibody (409.5.3) and in which the V.sub.H and V.sub.L domains of
A' define a lysozyme isotopic antibody (E5.2). In order to
facilitate purification of the desired diabody product, the gene
encoding V.sub.HB' and V.sub.LA' includes a hexahistidine tag,
whereas the gene encoding V.sub.HA' and V.sub.LB' does not.
[0014] FIG. 5. Line drawing representing the a-carbon trace of an
intact IgG1 (Protein Data Bank (pdb) entry 1IGY) (Harris et al.,
1998, Crystallographic structure of an intact IgG1 monoclonal
antibody, J. Mol. Biol. 275(5): 861-72). (For a description of the
Protein Data Bank (pdb), see Berman et al., 2000, The Protein Data
Bank, Nucl. Acids Res. 235-42; Saqi et al., 1994, PdbMotif--a tool
for the automatic identification and display of motifs in protein
structures, Comput. Appl. Biosci. 10(5): 545-46.) Thick lines
represent the heavy chains and thin lines represent the light
chains. The Fv and C.sub.H1 domains of the Fab fragment and the
C.sub.H2 and C.sub.H3 domains of the Fc fragment are labeled.
Ball-and-stick modeling, indicated by gray arrowheads, represent
disulfide cysteine bonds. Clusters of disulfide bridging
interactions occur in the flexible hinge region located between the
Fab and Fc fragments. These interactions may aid in dimerization
and provide structural integrity of this otherwise highly flexible
region in the immunoglobulin. Drawing was created with the program
SETOR (Evans, 1993, SETOR: Hardware lighted three-dimensional solid
model representations of macromolecules, J. Mol. Graphics, 11:
134-38).
[0015] FIG. 6. Line drawing representing the a-carbon trace of a
Fab fragment that can be used as the structural element for design
of an assembly unit (pdb entry 1CIC). The heavy lines represent the
heavy chain and the light lines represent the light chain. The
domains of the heavy chain (V.sub.H and C.sub.H1) and the light
chain (V.sub.L and C.sub.L) are labeled. Also indicated is the
flexible Fab "elbow" or bend region connecting the variable domains
and constant domains. The Fab angle of the bend varies considerably
(127-176.degree.) even among members of the same antibody
class.
[0016] FIG. 7. Schematic representation of various IgGs including
monovalent, bivalent, monospecific and bispecific antibodies. IgGs
that are derived from a single cell line are homozygous for IgG.
The resulting IgGs are therefore bivalent-monospecific antibodies.
A hybrid hybridoma, e.g., a quadroma, arises from a fusion cell
line. IgGs that are produced by hybrid hybridomas may be mixtures
of heterologous bivalent-bispecific (e.g.,
heterologous-F(ab').sub.2) and homozygous bivalent-monospecific
(e.g., F(ab').sub.2) IgG. Hybrid hybridoma heterodimers therefore
represent a source of bivalent-bispecific F(ab').sub.2. The intact
IgG molecules or binding derivative or binding fragment thereof can
be used as the basic elements for the design of assembly units.
[0017] FIG. 8. Schematic representation of an IgG molecule cleaved
into its component fragments, F(ab').sub.2 and Fc, upon limited
exposure to protease. The hinge region, containing several
disulfide-bond interactions, helps maintain dimerization of the Fab
fragments. Subsequent exposure of the F(ab').sub.2 to reducing
conditions disrupts the hinge disulfide bridging interactions
between the fragments to yield monomeric Fab. Separate functional
fragments of the IgG can be isolated (i.e., Fab fragments) for
specific uses in the design of assembly units such as creating
bivalent-bispecific heterologous F(ab').sub.2 by chemical
cross-linking.
[0018] FIGS. 9(A-D). Dimerization motifs that have been developed
to promote the multimerization of antigen-binding fragments that
contain various specificities. Leucine zipper motifs (depicted as
elongated ovals) such as Jun-Fos or GCN4 (Kostelny et al., 1992,
Formation of a bispecific antibody by the use of leucine zippers,
J. Immunol. 148(5): 1547-53; de Kruif et al., 1996, Leucine zipper,
dimerized bivalent and bispecific scFv antibodies from a
semi-synthetic antibody phage display library, J. Biol. Chem.
271(13): 7630-34), or four-helix bundle motifs (depicted as
rectangles in (C) and (D)), such as Rop (Pack et al., 1993,
Improved bivalent miniantibodies, with identical avidity as whole
antibodies, produced by high cell density fermentation of
Escherichia coli, Biotechnology (NY) 11(11): 1271-77; Muller et
al., 1998, A dimeric bispecific miniantibody combines two
specificities with avidity, FEBS Lett. 432(1-2): 45-49), may be
employed to promote the stable dimerization of antigen-binding
multimers. These dimerized antigen-binding multimers may be
utilized as the structural and joining elements in assembly unit
fabrication.
[0019] FIG. 10. Diagram of ROP protein, a four-helix bundle.
[0020] FIG. 11. Staged assembly of assembly units. In practice,
each step in the staged assembly will be carried out in a massively
parallel fashion. In step 1, an initiator unit is immobilized on a
solid substrate. In the embodiment of the invention illustrated
here, the initiator unit has a single joining element. In step 2, a
second assembly unit is added. The second unit has two
non-complementary joining elements, so that the units will not
self-associate in solution. One of the joining elements on the
second assembly unit is complementary to the joining element on the
initiator unit. Unbound assembly units are washed away between each
step (not shown).
[0021] After incubation, the second assembly unit binds to the
initiator unit, resulting in the formation of a nanostructure
intermediate made up of two assembly units. In step 3, a third
assembly unit is added. This unit has two non-complementary joining
elements, one of which is complementary to the only unpaired
joining element on the nanostructure intermediate. This unit also
has a functional unit ("F3").
[0022] A fourth assembly unit with functional element "F4" and a
fifth assembly unit with functional element "F5" are added in steps
4 and 5, respectively, in a manner exactly analogous to steps 2 and
3. In each case, the choice of joining elements prevents more than
one unit from being added at a time, and leads to a tightly
controlled assembly of functional units in pre-designated
positions.
[0023] FIG. 12. Generation of a nanostructure from subassemblies. A
nanostructure can be generated through the sequential addition of
subassemblies, using steps analogous to those used for the addition
of individual assembly units as illustrated above in FIG. 2. The
arrow indicates the addition of a subassembly to a growing
nanostructure.
[0024] FIG. 13. A diagram illustrating the addition of protein
units and inorganic elements to a nanostructure according to the
staged assembly methods of the invention. In step 1, an initiator
unit is bound to a solid substrate. In step 2, an assembly unit is
bound specifically to the initiator unit. In step 3, an additional
assembly unit is bound to the nanostructure undergoing assembly.
This assembly unit comprises an engineered binding site specific
for a particular inorganic element. In step 4, the inorganic
element (depicted as a cross-hatched oval) is added to the
structure and bound by the engineered binding site. Step 5 adds
another assembly unit with a binding site engineered for
specificity to a second type of inorganic element, and that second
inorganic element (depicted as a hatched diamond) is added in step
6.
[0025] FIG. 21. Diagram of eleven steps of a staged assembly that
utilizes four bispecific assembly units and one tetraspecific
assembly unit to make a two-dimensional nanostructure.
[0026] FIGS. 22(A-B). Diagram of a staged assembly that utilizes
nanostructure intermediates as subassemblies. In Steps 1-3, a
nanostructure intermediate is constructed, two joining elements are
capped and the nanostructure intermediate is released from the
solid substrate. In Step 5, the nanostructure intermediate from
Step 3 is added to an assembly intermediate (shown in Step 4
attached to the solid substrate) as an intact subassembly.
[0027] FIGS. 23(AA-BF). Diagram of the sequence of the 32 steps
used in the staged assembly of an exemplary cubic nanostructure.
The cubic nanostructure is assembled from assembly units comprising
structural elements from engineered diabody and triabody fragments.
The joining elements of the assembly units are the multispecific
binding domains from diabodies or triabodies. Seven complementary
joining pairs are used: A and A', B and B', C and C', D and D', E
and E', F and F', and G and G'. The numbering (1-32) indicates the
assembly unit added during each step.
DETAILED DESCRIPTION OF THE INVENTION
[0028] DEFINITIONS: The terms in this application are generally
used in a manner consistent with their ordinary meaning in the art.
To provide clarity, however, in the event of a disagreement in the
art, the following definitions control.
[0029] Antibody Assembly Unit: An assembly unit in which at least
one joining element, structural element or functional element is an
antibody or antibody fragment, or a binding derivative thereof. The
antibodies, binding derivatives or binding fragments may be of any
class of immunoglobulin molecules, including IgG, IgM, IgE, IgA,
IgD and any subclass thereof.
[0030] Antibody Fragment: A portion of an antibody with specific
binding affinity for an epitope. Examples of antibody fragments
include, without limitation, Fab or F(ab').sub.2 antibody
fragments, single-chain antibody fragments (scFvs), bispecific IgG,
chimeric IgG or bispecific heterodimeric F(ab').sub.2 antibodies,
diabodies or multimeric scFv fragments.
[0031] Assembly Unit: An assembly unit is an assemblage of atoms
and/or molecules comprising structural elements, joining elements
and/or functional elements. Preferably, an assembly unit is added
to a nanostructure as a single unit through the formation of
specific, non-covalent interactions. An assembly unit may comprise
two or more sub-assembly units. An assembly unit may comprise one
or more structural elements, and may further comprise one or more
functional elements and/or one or more joining elements. If an
assembly unit comprises a functional element, that functional
element may be attached to or incorporated within a joining element
or, in certain embodiments, a structural element. Such an assembly
unit, which may comprise a structural element and one or a
plurality of non-interacting joining elements, may be, in certain
embodiments, structurally rigid and have well-defined recognition
and binding properties.
[0032] Assembly Unit, Initiator: An initiator assembly unit is the
first assembly unit incorporated into a nanostructure that is
formed by the staged assembly method of the invention. It may be
attached, by covalent or non-covalent interactions, to a solid
substrate or other matrix as the first step in a staged assembly
process. An initiator assembly unit is also known as an "initiator
unit."
[0033] Binding Fragment, Binding Derivative: A binding derivative
of an antibody or antibody fragment is a derivative that exhibits
the binding specificity of the antibody, antibody fragment,
single-chain antibody fragment (scFv), etc., from which the binding
derivative is derived. A binding fragment of an antibody or
antibody fragment is a fragment that exhibits the binding
specificity of the antibody, antibody fragment, single-chain
antibody fragment (scFv), etc., from which the binding fragment is
derived.
[0034] Bottom-up: Bottom-up assembly of a structure (e.g.,a
nanostructure) is formation of the structure through the joining
together of substructures using, for example, self-assembly or
staged assembly.
[0035] Capping Unit: A capping unit is an assembly unit that
comprises at most one joining element. Additional assembly units
cannot be incorporated into the nanostructure through interactions
with the capping unit once the capping unit has been incorporated
into the nanostructure.
[0036] Derivative: Derivatives of a protein of interest used in the
methods of the invention, e.g., an antibody, can be made by
altering sequences by substitutions, additions or deletions that
provide for functionally equivalent molecules. Due to the
degeneracy of nucleotide coding sequences, other DNA sequences that
encode substantially the same amino acid sequence as the gene
encoding the protein of interest may be used in the practice of the
present invention. These include, but are not limited to,
nucleotide sequences comprising all or portions of a gene, which is
altered by the substitution of different codons that encode a
functionally equivalent amino acid residue within the sequence,
thus producing a silent change.
[0037] Likewise, derivatives of a protein of interest include, but
are not limited to, those containing, as a primary amino acid
sequence, all or part of the amino acid sequence of a protein of
interest including altered sequences in which functionally
equivalent amino acid residues are substituted for residues within
the sequence resulting in a silent change. For example, one or more
amino acid residues within the sequence can be substituted by
another amino acid of a similar polarity that acts as a functional
equivalent, resulting in a silent alteration. Substitutes for an
amino acid within the sequence may be selected from other members
of the class to which the amino acid belongs. For example, the
nonpolar (hydrophobic) amino acids include alanine, leucine,
isoleucine, valine, proline, phenylalanine, tryptophan and
methionine. The polar neutral amino acids include glycine, serine,
threonine, cysteine, tyrosine, asparagine, and glutamine. The
positively charged (basic) amino acids include arginine, lysine and
histidine. The negatively charged (acidic) amino acids include
aspartic acid and glutamic acid.
[0038] Alternatively, derivatives or analogs of antibodies include
but are not limited to those molecules comprising regions that are
substantially homologous to the antibody of interest or a binding
fragment thereof (e.g., in various embodiments, at least 60% or 70%
or 80% or 90% or 95% identity over an amino acid sequence of
identical size or when compared to an aligned sequence in which the
alignment is done by a computer homology program known in the art)
or whose encoding nucleic acid is capable of hybridizing to a
sequence encoding the protein of interest, under highly stringent
or moderately stringent conditions. Such highly or moderately
stringent conditions are commonly known in the art.
[0039] First Assembly Unit, First Element: For clarity, assembly
units or elements are sometimes referred to using labels such as
"first" or "second". This is purely a labeling convention and in no
way indicates the position of the referenced assembly unit or
element within the nanostructure.
[0040] Functional Element: A functional element is a moiety
exhibiting any desirable physical, chemical or biological property
that may be built into, bound or placed by specific covalent or
non-covalent interactions, at well-defined sites in a
nanostructure. Alternatively, a functional element can be used to
provide an attachment site for a moiety with a desirable physical,
chemical, or biological property. Examples of functional elements
include, without limitation, a peptide, protein (e.g., enzyme),
protein domain, small molecule, inorganic nanoparticle, atom,
cluster of atoms, magnetic, photonic or electronic nanoparticles,
or a marker such as a radioactive molecule, chromophore,
fluorophore, chemiluminescent molecule, or enzymatic marker. Such
functional elements can also be used for cross-linking linear,
one-dimensional nanostructures to form two-dimensional and
three-dimensional nanostructures.
[0041] Joining Element: A joining element is a portion of an
assembly unit that confers binding properties on the unit,
including, but not limited to: binding domain, hapten, antigen,
peptide, PNA, DNA, RNA, aptamer, polymer or other moiety, or
combination thereof, that can interact through specific,
non-covalent interactions, with another joining element.
[0042] Joining Elements, Complementary: Complementary joining
elements are two joining elements that interact with one another
through specific, non-covalent interactions.
[0043] Joining Elements, Non-Complementary: Non-complementary
joining elements are two joining elements that do not specifically
interact with one another, nor demonstrate any tendency to
specifically interact with one another.
[0044] Joining Pair: A joining pair is two complementary joining
elements.
[0045] Nanomaterial: A nanomaterial is a material made up of a
crystalline, partially crystalline or non-crystalline assemblage of
nanoparticles.
[0046] Nanoparticle: A nanoparticle is an assemblage of atoms or
molecules, bound together to form a structure with dimensions in
the nanometer range (1-1000 nm). The particle may be homogeneous or
heterogeneous. Nanoparticles that contain a single crystal domain
are also called nanocrystals.
[0047] Nanostructure or Nanodevice: A nanostructure or nanodevice
is an assemblage of atoms and/or molecules comprising assembly
units, i.e., structural, functional and/or joining elements, the
elements having at least one characteristic length (dimension) in
the nanometer range, in which the positions of the assembly units
relative to each other are established in a defined geometry. The
nanostructure or nanodevice may also have functional substituents
attached to it to provide specific functionality.
[0048] Nanostructure intermediate: A nanostructure intermediate is
an intermediate substructure created during the assembly of a
nanostructure to which additional assembly units can be added. In
the final step, the intermediate and the nanostructure are the
same.
[0049] Non-covalent Interaction, Specific: A specific non-covalent
interaction is, for example, an interaction that occurs between an
assembly unit and a nanostructure intermediate.
[0050] Protein: In this application, the term "protein" is used
generically to referred to peptides, polypeptides and proteins
comprising a plurality of amino acids, and is not intended to imply
any minimum number of amino acids.
[0051] Removing: Removing of unbound assembly is accomplished when
they are rendered unable to participate in further reactions with
the growing nanostructure, whether or not they are physically
removed.
[0052] Self-assembly: Self-assembly is spontaneous organization of
components into an ordered structure. Also known as
auto-assembly.
[0053] Staged Assembly of a Nanostructure: Staged assembly of a
nanostructure is a process for the assembly of a nanostructure
wherein a series of assembly units are added in a pre-designated
order, starting with an initiator unit that is typically
immobilized on a solid matrix or substrate. Each step results in
the creation of an intermediate substructure, referred to as the
nanostructure intermediate, to which additional assembly units can
then be added. An assembly step comprises (i) a linking step,
wherein an assembly unit is linked to an initiator unit or
nanostructure intermediate through the incubation of the matrix or
substrate with attached initiator unit or nanostructure
intermediate in a solution comprising the next assembly units to be
added; and (ii) a removal step, e.g., a washing step, in which
excess assembly units are removed from the proximity of the
intermediate structure or completed nanostructure. Staged assembly
continues by repeating steps (i) and (ii) until all of the assembly
units are incorporated into the nanostructure according to the
desired design of the nanostructure. Assembly units bind to the
initiator unit or nanostructure intermediate through the formation
of specific, non-covalent bonds. The joining elements of the
assembly units are chosen so that they attach only at
pre-designated sites on the nanostructure intermediate. The
geometry of the assembly units, the structural elements, and the
relative placement of joining elements and functional elements, and
the sequence by which assembly units are added to the nanostructure
are all designed so that functional units are placed at
pre-designated positions relative to one another in the structure,
thereby conferring a desired function on the completely assembled
nanostructure.
[0054] Structural Element: A structural element is a portion of an
assembly unit that provides a structural or geometric linkage
between joining elements, thereby providing a geometric linkage
between adjoining assembly units. Structural elements provide the
structural framework for the nanostructure of which they are a
part.
[0055] Subassembly: A subassembly is an assemblage of atoms or
molecules consisting of multiple assembly units bound together and
capable of being added as a whole to an assembly intermediate
(e.g., a nanostructure intermediate). In many embodiments of the
invention, structural elements also support the functional elements
in the assembly unit.
[0056] Top-down: Top-down assembly of a structure (e.g.,a
nanostructure) is formation of a structure through the processing
of a larger initial structure using, for example, lithographic
techniques.
[0057] Antibody Assembly Units
[0058] The present invention provides a new class of assembly units
that can be used in production of nanostructures. These "antibody
assembly units" contain at least one joining, structural or
functional element that is an antibody or antibody fragment. In
addition, the assembly unit may contain structural elements and/or
other joining and functional elements.
[0059] Chimeric Antibodies and Antibody Fragments
[0060] The present invention provides for the staged assembly of
nanostructures that utilizes assembly units comprising chimeric
antibodies or antibody fragments. The production of fusion or
chimeric protein products (comprising a desired protein (e.g., an
IgG), fragment, analog, or derivative joined via a peptide bond to
a heterologous protein sequence (of a different protein)). Such
chimeric protein products can be made by ligating the appropriate
nucleic acid sequences encoding the desired amino acid sequences to
each other by methods known in the art, in the proper reading
frame, and expressing the chimeric product by methods commonly
known in the art. Alternatively, such a chimeric product may be
made by protein synthetic techniques, e.g., by use of a peptide
synthesizer.
[0061] The three-dimensional structures of IgG and its binding
derivatives or binding fragments, e.g., IgG, Fab, scFv,
(scFv).sub.2 (scFv).sub.3), have been solved (Braden et al., 1996,
Crystal structure of an Fv-Fv idiotope-anti-idiotope complex at 1.9
.ANG. resolution, J. Mol. Biol. 264(1): 137-51; Ban et al., 1994,
Crystal structure of an idiotype-anti-idiotype Fab complex, Proc.
Natl. Acad. Sci. U.S.A. 91(5): 1604-08; Perisic et al., 1994,
Crystal structure of a diabody, a bivalent antibody fragment,
Structure 2(12): 1217-26; Harris et al., 1998, Crystallographic
structure of an intact IgG1 monoclonal antibody, J. Mol. Biol.
275(5): 861-72; Pei et al., 1997, The 2.0-A resolution crystal
structure of a trimeric antibody fragment with noncognate
V.sub.H-V.sub.L domain pairs shows a rearrangement of V.sub.H CDR3,
Proc. Natl. Acad. Sci. USA 94(18): 9637-42). Each IgG-derived
antibody fragment preferably contains at least one monovalent and
monospecific complementarity determining region (CDR) or joining
element. The CDR is preferably the site contained in each structure
at which the highly specific intermolecular interaction can occur
between the protein components.
[0062] Recombinantly engineered antibodies meet many of the basic
criteria for use in the construction of assembly units for
staged-assembly of nanostructures and are preferred sources of
joining elements used for fabricating such nanostructures. Not only
are such recombinant antibody binding domains structurally well
characterized, they also have inherent binding specificities
(joining elements) necessary for assembly unit addition.
[0063] For example, the known three-dimensional structure of many
recombinant engineered components can serve as a guide for design
of structural modifications to the antibody fragment that will
enable the insertion of peptides (for example, at the site of a
surface loop) that will confer novel binding, structural or
functional properties to the antibody fragment. Moreover, there is
a huge diversity of intermolecular specificities, such as that
involving an antibody and a specific epitope, that can be either
designed and constructed, or selected from a library. Advances in
recombinant antibody technology have led to the creation of
multivalent, multispecific and multifunctional antibodies
(Chaudhary et al., 1989, A recombinant immunotoxin consisting of
two antibody variable domains fused to Pseudomonas exotoxin, Nature
339(6223): 394-97; Neuberger et al. 1984, Recombinant antibodies
possessing novel effector functions, Nature 312(5995): 604-08;
Wallace et al., 2001, Exogenous antigen targeted to FcgammaRI on
myeloid cells is presented in association with MHC class I, J.
Immunol. Methods 248(1-2): 183-94) that may be used, according to
the methods of the invention, as sources of structural elements and
joining elements. Such multivalent, multispecific and
multifunctional antibodies can be modified by the addition of
functional groups for the construction of assembly units used for
the fabrication of nanostructures as described herein.
[0064] Antibody Joining Elements
[0065] Joining Elements Exhibiting Antigen-Antibody
Interactions
[0066] In certain embodiments of the invention, joining elements
are derived from antibodies, or binding derivatives or binding
fragments thereof, and exhibit antigen-antibody interactions which
are used in the formation of a joining pair. Structural information
is readily available for a variety of antibody-antigen complexes.
Such structural information may be used to design joining elements
for the fabrication of nanostructures according to the methods of
the invention. The variable domains of antibodies are designed to
interact with specificity to an antigenic target. Their structure
and stability are well-characterized in the art, and antibodies and
antibody binding fragments may be engineered using methods well
known in the art. Consequently, the variable domains of antibodies
represent a class of molecules with great potential as joining
elements for use as nanostructure assembly units. Such elements
provide the basis for specific binding interactions between
assembly units and initiators or nanostructure intermediates and
are described herein.
[0067] It is well known in the art that binding of antibody to
antigen is highly specific (Davies et al., 1990, Antibody-antigen
complexes, Ann. Rev. Biochem. 59: 439-73; Mian et al., 1991,
Structure, function and properties of antibody binding sites, J.
Mol. Biol. 217(1): 133-51; Wilson et al., 1994, Antibody-antigen
interactions: new structures and new conformational changes, Curr.
Opin. Struct. Biol. 4(6): 857-67; Davies et al., 1996, Interactions
of protein antigens with antibodies, Proc. Natl. Acad. Sci. USA
93(1): 7-12). This high specificity has been shown to correlate
with the high complementarity between the antibody combining site
and the antigenic determinant, i.e., the epitope or hapten. This
complementarity is defined by the antibody determinant face,
defined as the complementarity determining region (CDR) and the
antigenic determinant surface, which are in contact, so that the
depressions in one are filled by the protrusions from the other.
Complementarity also exists by physical and chemical properties
such as opposed, oppositely charged side-chain interactions that
form ionic bonds. The specificity occurring between the CDR and the
antigenic determinant surface can define one type or pair of
non-complementary joining element interactions.
[0068] Many aromatic side-chain residues, forming hydrophobic
interactions, are present in these antibody-antigen interactions.
Complementarity between some antigen-antibody complexes is so
precise that even water molecules are excluded access from the
interface. This particular feature, along with the structural and
chemical diversity of the residues within in the CDR loop,
including the insertions and deletions, permit specificity and
diversity of ligand binding by different antibodies (Winter et al.,
1991, Man-made antibodies, Nature 349(6307): 293-99; Davies et al.,
1996, Interactions of protein antigens with antibodies, Proc. Natl.
Acad. Sci. USA 93(1): 7-12); Wedemayer et al., 1997, Structural
insights into the evolution of an antibody combining site, Science
276(5319): 1665-69). Such known specificity and diversity of ligand
binding by different antibodies can be used in designing joining
elements for use in constructing nanostructures according to the
methods of the invention.
[0069] Antibodies or portions thereof used in the methods of the
invention can be multispecific (i.e., demonstrate binding affinity
towards more than one ligand) or monospecific (i.e., demonstrate
binding affinity towards only one ligand). In general, antibodies
demonstrate binding affinity in the 10.sup.-1 to 10.sup.-4 nM range
or better (Padlan, 1994, Anatomy of the antibody molecule, Mol.
Immunol. 31(3): 169-217).
[0070] An immunoglobulin light or heavy chain variable region
consists of a "framework" region interrupted by three hypervariable
regions, the CDRs. The Fv fragment contains six variable loop
regions, three from the V.sub.L chain and three from the V.sub.H
chain. Each of the variable polypeptide loop regions contained in
the variable chains display variability in residue sequence and
length. Residues within this region are assigned either to
hypervariable, complementarity-determining-regions (CDRs) or to
non-hypervariable or framework regions (Wu et al., 1970, An
analysis of the sequences of the variable regions of Bence Jones
proteins and myeloma light chains and their implications for
antibody complementarity, J. Exp. Med 132(2): 211-50; Wu et al.,
1975, Similarities among hypervariable segments of immunoglobulin
chains, Proc. Natl. Acad. Sci. USA 72(12): 5107-10; Wu et al, 1993,
Length distribution of CDRH3 in antibodies, Proteins 16(1): 1-7).
The extent of the framework region and CDRs has been precisely
defined (see Kabat et al., 1983, Sequences of Proteins of
Immunological Interest, U.S. Department of Health and Human
Services).
[0071] Together, these variable loop regions define, almost
entirely, the antigen-recognition site of the antibody. Both CDR3s
(CDR3-L and CDR3-H) are the most prominent in antibody-antigen
recognition interactions and are the most variable in sequence and
conformation. The contributions from the CDR loops from both the
V.sub.L and the V.sub.H chains on binding to antigen are relatively
consistent. Structural analyses of antibodies complexed with
antigen have determined that approximately 41-44% of the
interacting surface area is contributed by the light chain with the
heavy chain contributing 56-59% (Davies et al, 1990,
Antibody-antigen complexes, Annu. Rev. Biochem. 59: 439-73). The
overall number of residues that interact with the antigen is rather
small. Structural analysis of antibody-antigen complexes have
revealed that, on average, only 15 antibody residues interact with
antigen. Other residues within the CDR loops, however, may offer
additional antibody-antigen interactions, as well as provide a
structural role in order to maintain the antibody combining site
structure ((Davies et al., 1990, Antibody-antigen complexes, Annu.
Rev. Biochem. 59: 439-73; Wilson and Stanfield, 1994,
Antibody-antigen interactions: new structures and new
conformational changes, Curr. Opin. Struct. Biol. 4(6): 857-67;
Davies and Cohen, 1996, Interactions of protein antigens with
antibodies, Proc. Natl. Acad. Sci. USA 93(1): 7-12).
[0072] Joining Elements Comprising a Recombinantly Engineered
Antibody or Binding Derivative or Binding Fragment Thereof
[0073] In certain embodiments of the invention, a joining element
comprises a recombinantly engineered antibody or binding derivative
or binding fragment thereof. There are many examples of
recombinantly engineered antibodies known in the art that are
multivalent, multispecific and/or multifunctional, and that are
suitable as joining elements for use in the design of assembly
units for staged assembly of nanostructures. Such assembly units
may either be unmodified or be modified as described herein, for
use in the methods of the invention for fabrication of a desired
nanostructure.
[0074] Some examples of recombinantly engineered antibodies, or
binding derivatives or binding fragments thereof, for use as
joining elements include, but are not limited to:
[0075] (i) immunoglobulins from any class including IgG, IgM, IgE,
IgA, IgD or any subclass thereof, including immunoglobulins derived
from a hybrid hybridoma or from a quadroma (which is a cell line
that produces a particular bispecific antibody, i.e. an antibody
molecule with two different Fab binding segments);
[0076] (ii) monovalent and monospecific antibodies such as Fv, scFv
and Fab (Ban, et al., 1994, Crystal structure of an
idiotype-anti-idiotype Fab complex, Proc. Natl. Acad. Sci. USA
91(5): 1604-08, Freund et al, 1994, Structural and dynamic
properties of the Fv fragment and the single-chain Fv fragment of
an antibody in solution investigated by heteronuclear
three-dimensional NMR spectroscopy, Biochemistry 33(11): 3296-303;
Boulot et al., 1990, Crystallization and preliminary X-ray
diffraction study of the bacterially expressed Fv from the
monoclonal anti-lysozyme antibody D1.3 and of its complex with the
antigen, lysozyme, J. Mol. Biol. 213(4): 617-19; Padlan, 1994,
Anatomy of the antibody molecule, Mol. Immunol. 31(3):
169-217);
[0077] (iii) bivalent, trivalent, mono-, bi-, or tri-specific
antibodies with or without added functionalities, such as IgGs
derived from hybrid hybridomas, F(ab').sub.2, diabodies,
triabodies, tetrabodies, heterologous-F(ab').sub.2, Fab-scFv
fusions or F(ab').sub.2-scFv fusions (Milstein and Cuello, 1983,
Hybrid hybridomas and their use in immunohistochemistry, Nature
305(5934): 537-40; Neuberger et al., 1984, Recombinant antibodies
possessing novel effector functions, Nature 312(5995): 604-08;
Weiner, 1992, Bispecific IgG and IL-2 therapy of a syngeneic B-cell
lymphoma in immunocompetent mice, Int. J. Cancer Suppl. 7: 63-66,
Holliger and Winter, 1993, Engineering bispecific antibodies, Curr.
Opin. Biotechnol. 4(4): 446-49; Dolezal et al., 1995, Escherichia
coli expression of a bifunctional Fab-peptide epitope reagent for
the rapid diagnosis of HIV-1 and HIV-2, Immunotechnology 1(3-4):
197-209; Tso et al., 1995, Preparation of a bispecific F(ab').sub.2
targeted to the human IL-2 receptor, J. Hematother. 4(5): 389-94;
Atwell et al., 1996, Design and expression of a stable bispecific
scFv dimer with affinity for both glycophorin and N9 neuraminidase,
Mol. Immunol. 33(17-18): 1301-12; de Kruif et al., 1996, Leucine
zipper dimerized bivalent and bispecific scFv antibodies from a
semi-synthetic antibody phage display library, J. Biol. Chem.
271(13): 7630-34; Kipriyanov et al., 1998, Bispecific
CD3.times.CD19 diabody for T cell-mediated lysis of malignant human
B cells, hit. J. Cancer 77(5): 763-72; Muller et al., 1998, A
dimeric bispecific miniantibody combines two specificities with
avidity, FEBS Lett. 432(1-2): 45-49; Carter 2001, Bispecific human
IgG by design, J. Immunol. Methods 248(1-2): 7-15; (Fell et al.,
1991, Genetic construction and characterization of a fusion protein
consisting of a chimeric F(ab') with specificity for carcinomas and
human IL-2, J. Immunol. 146(7): 2446-52; Iliades et al., 1997,
Triabodies: single chain Fv fragments without a linker form
trivalent trimers, FEBS Lett. 409(3): 437-41; Hudson and Kortt,
1999, High avidity scFv multimers; diabodies and triabodies, J.
Immunol. Methods 231(1-2): 177-89; Schoonjans et al, 2000,
Efficient heterodimerization of recombinant bi- and trispecific
antibodies, Bioseparation 9(3): 179-83; Schoonjans et al., 2000,
Fab chains as an efficient heterodimerization scaffold for the
production of recombinant bispecific and trispecific antibody
derivatives, J. Immunol. 165(12): 7050-57);
[0078] (iv) tetravalent antibodies that are either, mono-, bi-,
tri- or tetraspecific antibodies, with or without added
functionalities, such as tetrabodies, Ig-G binding derivative-scFv
fusions or IgG-scFv fusions (Pack et al., 1995, Tetravalent
miniantibodies with high avidity assembling in Escherichia coli, J.
Mol. Biol. 246(1): 28-34, Coloma and Morrison, 1997, Design and
production of novel tetravalent bispecific antibodies, Nat.
Biotechnol. 15(2): 159-63; Alt et al., 1999, Novel tetravalent and
bispecific IgG-like antibody molecules combining single-chain
diabodies with the immunoglobulin gamma1 Fc or CH3 region, FEBS
Lett. 454(1-2): 90-4; Le Gall et al., 1999, Di-, tri- and
tetrameric single chain Fv antibody fragments against human CD19:
effect of valency on cell binding, FEBS Lett. 453(1-2): 164-68;
Santos et al., 1999, Generation and characterization of a single
gene-encoded single-chain-tetravalent antitumor antibody, Clin.
Cancer Res. 5(10 Suppl): 3118s-3123s; Goel et al., 2000,
Genetically engineered tetravalent single-chain Fv of the
pancarcinoma monoclonal antibody CC49: improved biodistribution and
potential for therapeutic application, Cancer Res. 60(24): 6964-71;
Todorovska et al., 2001, Design and application of diabodies,
triabodies and tetrabodies for cancer targeting, J. Immunol.
Methods 248(1-2): 47-66); and
[0079] (v) fusions of an scFv and a binding derivative of an IgG
(see, e.g., Huston et al., 1991, Protein engineering of
single-chain Fv analogs and fusion proteins, Methods Enzymol. 203:
46-88); fusions of a cytokine and a binding derivative of an IgG
(wherein the cytokine is, e.g., a BCDF (B-cell differentiation
factor), a BCGF (B-cell growth factor), a motogenic cytokine, a
chemotactic cytokine or chemokine, a CSF (colony stimulating
factor), an angiogenesis factor, a TRF (T-cell replacing factor),
an ADF (adult T-cell leukemia-derived factor), a PD-ECGF
(platelet-derived endothelial cell growth factor), a neuroleukin,
an interleukin, a lymphokine, a monokine, an interferon, etc.)(see,
e.g., Penichet and Morrison, 2001, Antibody-cytokine fusion
proteins for the therapy of cancer, J. Immunol. Methods 248(1-2):
91-101; Penichet et al., 1998, An IgG3-IL-2 fusion protein
recognizing a murine B cell lymphoma exhibits effective tumor
imaging and antitumor activity, J. Interferon Cytokine Res. 18(8):
597-607; Fell et al., 1991, Genetic construction and
characterization of a fusion protein consisting of a chimeric
F(ab') with specificity for carcinomas and human IL-2, J. Immunol.
146(7): 2446-52); fusions of a scFv and a leucine zipper (de Kruif
and Logtenberg, 1996, Leucine zipper dimerized bivalent and
bispecific scFv antibodies from a semi-synthetic antibody phage
display library, J. Biol. Chem. 271(13): 7630-34; see also Section
5.5.3); and fusions of a scFv and a Rop protein (see, e.g., Huston
et al., 1991, Protein engineering of single-chain Fv analogs and
fusion proteins, Methods Enzymol. 203: 46-88; see also Section
5.5.4).
[0080] Joining Elements Exhibiting Idiotope/Anti-Idiotope
Interactions
[0081] In certain embodiments of the invention,
idiotope/anti-idiotope interactions are used to design joining
elements for the construction of nanostructures according to the
methods of the invention. Since antibodies can recognize virtually
any antigen, they have the ability to recognize other antigenic
determinants contained on other antibodies. The immune responses
that arise from the potential antigenic determinants on antibodies
are called "idiotopic" (Jerne, 1974, Towards a network theory of
the immune system, Ann. Immunol. (Paris) 125C(1-2): 373-89; Davie
et al., 1986, Structural correlates of idiotopes, Annu. Rev.
Immunol. 4: 147-65). Idiotopes are the antigenic determinants
unique to a particular antibody or group of antibodies. Antibodies
bearing idiotopes can react with antibodies that recognize the
idiotope as antigen and are therefore termed "anti-idiotopic"
antibodies. In most cases, the idiotope has been shown by
immunological and structural techniques to associate partially or
entirely with the CDR of a specific mAb (FIG. 2). Idiotopic
antibodies are known to have as great or greater affinity toward
their specific anti-idiotopic antibody as toward their specific
antigen (Braden et al., 1996, Crystal structure of an Fv-Fv
idiotope-anti-idiotope complex at 1.9 .ANG. resolution, J. Mol.
Biol. 264(1): 137-51).
[0082] In some cases, the CDR anti-idiotope adopts a structural
conformation of an "internal-image" of the external antigen
(Bentley et al., 1990, Three-dimensional structure of an
idiotope-anti-idiotope complex, Nature 348(6298): 254-57; Ban et
al., 1994, Crystal structure of an idiotope-anti-idiotope Fab
complex, Proc. Natl. Acad. Sci. USA 91(5): 1604-08; Poljak, 1994,
An idiotope--anti-idiotope complex and the structural basis of
molecular mimicking, Proc. Natl. Acad. Sci. USA 91(5): 1599-1600;
Braden et al., 1996, Crystal structure of an Fv-Fv
idiotope-anti-idiotope complex at 1.9 .ANG. resolution, J. Mol.
Biol. 264(1): 137-51; Iliades et al., 1998, Single-chain Fv of
anti-idiotype 11-1G10 antibody interacts with antibody NC41
single-chain Fv with a higher affinity than the affinity for the
interaction of the parent Fab fragments, J. Protein Chem. 17(3):
245-54). In certain embodiments, idiotopic antibodies are used that
have equal or greater affinity towards antigen as anti-idiotopic
antibody (Braden et al., 1996, Crystal structure of an Fv-Fv
idiotope-anti-idiotope complex at 1.9 .ANG. resolution, J. Mol.
Biol. 264(1): 137-51, and references cited therein).
[0083] For example, antibodies that bind to a peptide of interest
and competitively inhibit the binding of the peptide to its
receptor can be used to generate anti-idiotope antibodies that
"mimic" the peptide receptor and, therefore, bind the peptide.
Anti-idiotope antibodies may be generated using techniques well
known to those skilled in the art (see, e.g., Greenspan and Bona,
1993, Idiotypes: structure and immunogenicity, FASEB J. 7(5):
437-44; and Nissinoff, 1991, Idiotypes: concepts and applications,
J. Immunol. 147(8): 2429-38).
[0084] Illustrative, non-limiting examples of
idiotope/anti-idiotope binding pairs useful in the compositions of
joining elements and methods of the present invention are provided
below in Table 1.
1TABLE 1 Idiotope/Anti-Idiotope Interactions Idiotope/Anti-Idiotope
Complex Reference Idiotope-Anti-Idiotope Fab-Fab Complex; Bentley
et al., 1990, Three-dimensional D1.3-E225 (Mus musculus) structure
of an idiotope-anti-idiotope complex, Nature 348(6298): 254-57
Idiotopic Antibody D1.3 Fv Braden et al., 1996, Crystal structure
of an Fragment-Anti-idiotopic Antibody E5.2 Fv Fv-Fv
idiotope-anti-idiotope complex at Fragment Complex (Mus musculus)
1.9 .ANG. resolution, J. Mol Biol. 264(1): 137-51 Fab of YsT9.1
(Ab1) and the Fab of its Evans et al. 1994, Exploring the mimicry
anti-idiotopic monoclonal antibody of polysaccharide antigens by
anti-idiotypic T91AJ5 (Ab2) antibodies. The crystallization,
molecular replacement, and refinement to 2.8 .ANG. resolution of an
idiotope-anti-idiotope Fab complex and of the unliganded
anti-idiotope Fab, J. Mol. Biol. 241(5): 691-705
Idiotope-Anti-idiotope complex of Poljak, 1994, An
idiotope-anti-idiotope antibody fragments complex and the
structural basis of molecular mimicking, Proc. Natl. Acad. Sci. USA
91(5): 1599-600 Fab fragment of the mouse Ban et al., 1996, Crystal
structure of an anti-anti-idiotypic monoclonal antibody
anti-anti-idiotype shows it to be (mAb) GH1002 self-complementary,
J. Mol. Biol. 255(4): 617-27 Anti-idiotopic Fab 409.5.3, made
against Ban et al., 1995, Structure of an an E2 specific feline
infectious peritonitis anti-idiotypic Fab against feline
peritonitis virus-neutralizing antibody 730.1.4 virus-neutralizing
antibody and a comparison with the complexed Fab, FASEB J. 9(1):
107-14
[0085] In certain embodiments, specific idiotope/anti-idiotope
intermolecular interactions are used as the joining elements to
link assembly units together in the staged assembly of a
nanostructure (FIG. 1). Each derived assembly unit is designed to
contain two specific idiotope/anti-idiotope binding surfaces that
are non-cross-reacting. This provides a means of creating a system
for the staged assembly of assembly units to form complex
nanostructures comprising various and diverse functional elements.
Multiple joining pairs can be created by standard methods of phage
display (Winter et al., 1994, Making antibodies by phage display
technology, Ann. Rev. Immunol. 12: 433-55;Viti et al., 2000, Design
and use of phage display libraries for the selection of antibodies
and enzymes, Methods Enzymol. 326: 480-505). Furthermore, the
three-dimensional structure of antibodies and antibody derivatives
are well-characterized (see, e.g., Braden et al. 1996, Crystal
structure of an Fv-Fv idiotope-anti-idiotope complex at 1.9 .ANG.
resolution, J. Mol. Biol. 264(1): 137-51; Ban et al., 1994, Crystal
structure of an idiotype-anti-idiotype Fab complex, Proc. Natl.
Acad. Sci. USA 91(5): 1604-08; Perisic et al. 1994, Crystal
structure of a diabody, a bivalent antibody fragment, Structure
2(12): 1217-26; Harris et al., 1998, Crystallographic structure of
an intact IgG1 monoclonal antibody, J. Mol. Biol. 275(5): 861-72;
Pei et al., 1997, The 2.0-.ANG. resolution crystal structure of a
trimeric antibody fragment with noncognate V.sub.H-V.sub.L domain
pairs shows a rearrangement of V.sub.H CDR3, Proc. Natl. Acad. Sci.
USA 94(18): 9637-42) and positions for engineering additional
functional elements may be identified by visual investigation of
the available X-ray coordinates.
[0086] In certain embodiments, one of the CDR domains (i.e., one of
the joining elements) of an antibody-derived assembly unit can be
engineered as an idiotope. The other CDR can be engineered as a
non-complementary anti-idiotope joining element. Since the joining
elements are non-identical and non-interactive with each other,
this design prevents self-polymerization of the protein component.
Such joining elements can be fabricated using combinations of
molecular biology and phage display technologies (Winter et al,
1994, Making antibodies by phage display technology, Ann. Rev.
Immunol. 12: 433-55; Viti et al., 2000, Design and use of phage
display libraries for the selection of antibodies and enzymes,
Methods Enzymol. 326: 480-505). The resulting antibody-derived
assembly unit will contain both an idiotopic CDR or joining element
and a non-complementary anti-idiotopic CDR joining element.
[0087] In certain embodiments of the invention, the assembly unit
to be coupled in the next addition cycle can be designed in an
analogous fashion, with a joining element that is an idiotope and a
joining element that is a non-complementary anti-idiotope. One CDR
of this assembly unit, however, can be engineered to associate with
one of the previous CDR components that functions as joining
elements. Therefore, in certain embodiments, the CDRs of two
adjacent assembly units can be designed to have joining elements
that have complementary idiotope/anti-idiotope interactions. Using
assembly units of this design allows for a defined directionality
or orientation of the linked assembly unit and of the staged
assembly as a whole, i.e., vectorial addition of each assembly
unit. Since the CDRs of diabodies are geometrically opposed, the
assembly units can be added to an initiator or nanostructure
intermediate in known orientation and direction.
[0088] Joining Elements Comprising Two Non-Complementary
Idiotopes
[0089] In certain embodiments, an assembly unit is fabricated that
comprises a diabody unit, wherein the non-complementary joining
elements are comprised of two non-complementary idiotopes.
[0090] A diabody, or a binding derivative or binding fragment
thereof, may be incorporated into a nanostructure in such a way
that only one of the two CDRs is used. In certain embodiments, the
CDRs themselves serve as joining elements, and the body of the
diabody between the two CDRs serves as a structural element.
[0091] Bispecific diabodies are derived from two non-paired scFv
fragments. The first portion of the hybrid fragment contains the
V.sub.H coding region from one Fv antibody and the second portion
contains the V.sub.L coding region derived from another Fv
antibody. The resulting V.sub.H-V.sub.L hybrid fragment is joined
together by a short Gly.sub.4Ser linker. The second hybrid fragment
will contain linkage of the analogous but opposite coding region
pair also joined together by a short Gly.sub.4Ser linker (FIGS. 2
and 3). The set of hybrid scFv fragments pair by intermolecular
interactions between the V.sub.H and V.sub.L domains.
[0092] In a specific embodiment illustrated in FIG. 4, the genes
used to create a first assembly unit ("Diabody Unit 1") are derived
from the lysozyme idiotopic antibody D1.3 (represented as V.sub.HA
and V.sub.LA in FIG. 4A) (Braden et al., 1996, Crystal structure of
an Fv-Fv idiotope-anti-idiotope complex at 1.9 .ANG. resolution, J.
Mol. Biol. 264(1): 137-51) and the feline infectious peritonitis
virus-neutralizing idiotopic antibody 730.1.4 (represented as
V.sub.HB and V.sub.LB in FIG. 4A) (Ban et al., 1994, Crystal
structure of an idiotype-anti-idiotype Fab complex, Proc. Natl.
Acad. Sci. USA 91(5): 1 604-08). The linker sequences joining the
hybrid V.sub.HA and V.sub.LB units and the hybrid V.sub.HB and
V.sub.LA units are designed based on those published by Huston et
al. (1988, Protein engineering of antibody binding sites: recovery
of specific activity in an anti-digoxin single-chain Fv analogue
produced in Escherichia coli, Proc. Natl. Acad. Sci. USA 85(16):
5879-83). The construct of Diabody Unit 1 is represented as
A.times.B in FIG. 4A. The locations of the promoter (p), ribosome
binding site (rbs), pelB leader (pelB), HSV and histidine (his)
tags and stop codons (Stop) are also indicated in FIG. 4. The
vector system used to engineer the diabody is pET25b (Novagen),
which contains a T7 promoter, ribosome binding site, pelB leader
sequence, HSV and His tag sequences.
[0093] FIG. 4B illustrates a second assembly unit (Diabody Unit 2)
comprises a diabody, wherein the non-complementary joining elements
are designed to contain two non-complementary anti-idiotopes. The
genes used to create this second assembly unit are derived from the
lysozyme anti-idiotopic antibody E5.2 (represented as V.sub.HA' and
V.sub.LA' in FIG. 4B) (Braden et al., 1996, Crystal structure of an
Fv-Fv idiotope-anti-idiotope complex at 1.9 .ANG. resolution, J.
Mol. Biol. 264(1): 137-51) and the feline infectious peritonitis
virus-neutralizing anti-idiotopic antibody 409.5.3 (represented as
V.sub.HB' and V.sub.LB' in FIG. 4B) (Ban et al., 1994, Crystal
structure of an idiotype-anti-idiotype Fab complex, Proc. Natl.
Acad. Sci. USA 91(5): 1 604-08). The construct of Diabody Unit 2 is
represented as A'.times.B'. These two exemplary assembly units can
be used in conjunction with an initiator unit to fabricate a
nanostructure by the methods of staged assembly described
herein.
[0094] Joining Elements Comprising a Peptide Epitope
[0095] In certain embodiments of the invention, joining elements
comprise peptide epitopes. Peptide epitopes may be engineered into
assembly units to act as joining elements that form a complementary
pair with an antibody or antibody binding fragment, the CDR of
which binds to the peptide epitope with specificity. Peptide
epitopes can be spliced into multiple defined regions contained
within the assembly units described above. Peptides epitopes are
particularly preferred as joining elements for use in a number of
embodiments, in addition to those embodiments wherein the peptide
epitope is used for cross-linking assembly units of adjacent
nanostructures together. Therefore, peptide epitopes provide
versatility to assembly units into which they are incorporated.
[0096] For example, in certain embodiments, peptide epitopes can
serve as joining elements for junctions that can be initiation
points for the assembly of new branches of a nanostructure from a
pre-existing branch. Such branching may be used to generate one,-
two- or three-dimensional structures. It may be used to expand
beyond a simple one-dimensional structure or to attach functional
units to a one-dimensional structure. Alternatively, such joining
elements can serve as the binding sites for the addition of
separately-fabricated nanostructure sub-assemblies to nanostructure
intermediates. In other embodiments, they can serve as binding
sites for antibodies that have linked or bound functional
elements.
[0097] In certain embodiments, assembly units comprise antibody
fragments that comprise peptide epitope joining elements. The
inherent flexibility within the Fab fragment may be used
advantageously for insertion of a joining element that enables
various cross-linked geometries between assembly units of
nanostructures in a staged assembly. In one embodiment, to
incorporate the additional intermolecular binding site on the Fab
fragment needed for staged assembly, the C-terminal distal end, or
the .beta.-turn regions, are engineered to contain a peptide
epitope. Exemplary peptide epitopes are set forth in Table 2.
[0098] Specific exemplary assembly units are variants of
bacteriophage T4 tail fiber protein gp37 in which the C-terminal
domain of the polypeptide is modified to include sequences that
confer specific binding properties on the entire molecule, e.g.,
sequences derived from avidin that recognize biotin, sequences
derived from immunoglobulin heavy chain that recognize
Staphylococcal A protein, sequences derived from the Fab portion of
the heavy chain of monoclonal antibodies to which their respective
Fab light chain counterparts could attach and form an
antigen-binding site, immunoactive sequences that recognize
specific antibodies, or sequences that bind specific metal ions.
These ligands may be immobilized to facilitate purification and/or
assembly.
2TABLE 2 Examples of Peptide Epitopes for Use as Joining Elements
Antibody/Antigenic-Peptide Sequence Reference (Antibody 8F5)
Complexed VKAETRLNPDLQPTE Tormo et al., 1994, Crystal With Peptide
From Human (SEQ ID NO: 1) structure of a human Rhinovirus (Serotype
2) rhinovirus neutralizing Viral Capsid Protein Vp2 antibody
complexed with a (Residues 156-170) peptide derived from viral
capsid protein VP2, EMBO J. 13(10): 2247-56 Fab59. complexed with a
YNKRKRIHIGPGRXFYT Ghiara et al., 1997, peptide mimic of the HIV-1
TKNIIGC Structure-based design of a V3 loop neutralization site.
(SEQ ID NO: 2) constrained peptide mimic of the HIV-1 V3 loop
neutralization site, J. Mol. Biol. 266(1): 31-39 Antibody
Campath-1H Fab/ GTSSPSAD James et al., 1999, 1.9 .ANG. Peptide
Antigen (SEQ ID NO: 3) structure of the therapeutic antibody
CAMPATH-1H Fab in complex with a synthetic peptide antigen. J. Mol.
Biol. 289(2): 293-301 Anti-Prion Fab 3F4 In APKTNMKHMA Kanyo et
al., 1999, Complex With Its Peptide (SEQ ID NO: 4) Antibody binding
defines a Epitope structure for an epitope that participates in the
PrPC-->PrPSc conformational change. J. Mol. Biol. 293(4): 855-63
Fab Fragment Monoclonal YTTSTRGDLAHVTTT Ochoa et al. 2000, A
Antibody 4C4 w/ (SEQ ID NO: 5) multiply substituted G-H Fmdv.
peptide loop from foot-and-mouth disease virus in complex with a
neutralizing antibody: a role for water molecules. J. Gen. Virol.
81 (Pt 6): 1495-505 Igg2A Fab (C3) Poliovirus CVTIMTVDNPASTTNK Wien
et al., 1995, Structure Type 1 Fragment DK of the complex between
the (SEQ ID NO: 6) Fab fragment of a neutralizing antibody for type
1 poliovirus and its viral epitope. Nat. Struct. Biol. 2(3): 232-43
Antibody Sm3 Complex TSAPDTRPAPGST Dokurno et al., 1998, With Its
Peptide Epitope (SEQ ID NO: 7) Crystal structure at 1.95 .ANG.
resolution of the breast tumour-specific antibody SM3 complexed
with its peptide epitope reveals novel hypervariable loop
recognition, J. Mol. Biol. 284(3): 713-28 Fab 58.2 Complex With
HIGPGRAFGG G Stanfield et al., 1999, Dual 12-Residue Cyclic Peptide
(SEQ ID NO: 8) conformations for the HIV-1 gp120 V3 loop in
complexes with different neutralizing Fabs, Structure Fold. Des.
7(2): 131-42 Monoclonal Antibody MSLPGRWKPK Lescar et al., 1997,
F11.2.32; Fab; complexed (SEQ ID NO: 9) Three-dimensional structure
with Hiv-1 Protease Peptide; of an Fab-peptide complex: structural
basis of HIV-1 protease inhibition by a monoclonal antibody, J.
Mol. Biol 267(5): 1207-22 Mn12H2 Igg2A Fab KDTNNNL van den Elsen et
al., 1997, Fragment; complexed with (SEQ ID NO: 10) Bactericidal
antibody Fluorescein-Conjugated recognition of a PorA Peptide
epitope of Neisseria meningitidis: crystal structure of a Fab
fragment in complex with a fluorescein-conjugated peptide, Proteins
29(1): 113-25
[0099] In one embodiment, a peptide epitope can replace the defined
.beta.-turn motifs contained in the fragment directly.
Alternatively, a peptide epitope can be linked to the C-terminal
amino acid of the CH1 heavy chain (Wallace et al., 2001, Exogenous
antigen targeted to FcgammaRI on myeloid cells is presented in
association with MHC class I, J. Immunol. Methods 248(1-2): 183-94)
by standard methods of molecular biology. Table 3 sets forth
examples of identified peptide regions contained in IgG and IgG
derivations that are suitable for insertion of joining elements or
functional elements.
3TABLE 3 Identified Peptide Regions Contained in IgG and IgG
Derivatives for Insertion of Joining Elements or Functional
Elements Residue Domain Secondary Structure (Chain).sup.2 IgG1
(Fc).sup.1 C.sub.H2 .beta.-turn res 265-269 res 295-299 res 311-317
(B, D) C.sub.H3 .beta.-turn res 408-414 res 449-452 res 464-466 (B,
D) C.sub.H3 C-terminal res 474 .alpha. C (B, D) Fab Fragment.sup.3
Fv .beta.-turn res 14-18 (A) res 11-16 (B) Fab Extended res 107-111
Bend Loop (A) Region res 115-120 (B) C.sub.H1 .beta.-turn res
149-153 res 198-202 (A) res 159-162 res 203-207 (B) C.sub.H1
C-terminal res 214 .alpha. C (A) res 217 (B) scFv.sup.4 V.sub.H
.beta.-turn res 13-16 res 88-90 res 40-43 (D) V.sub.L .beta.-turn
res 12-16 res 45-48 (C) V.sub.H C-terminal res 218 .alpha. C (D)
Diabody.sup.5 V.sub.H .beta.-turn res 13-16 res 39-44 res 62-66 res
73, 77 (A, C) V.sub.L C-terminal res 312 .alpha. C (A, C) Table 3
Notes: .sup.1Residue regions are defined in the Fc fragment of the
intact IgG1 from analysis of the atomic coordinates and numbered
according to the residue assignments deposited under entry 1IGY at
the Brookhaven National Laboratory protein data bank (BNL-pdb)
(Berman et al., 2000, The Protein Data Bank, Nucl. Acids Res.
235-42; 1977, The Protein Data Bank. A computer-based archival file
for macromolecular structures, Eur. J. Biochem. 80(2): 3 19-24).
.sup.2Chain assignments are labeled in accord with the
corresponding deposited pdb coordinates. .sup.3Resid+E,graue
regions are defined in the Fab fragment from analysis of the atomic
coordinates and numbered according to the residue assignments
deposited under entry 1CIC at the BNL-pdb. .sup.4Residue regions
are defined within the scFv fragment from analysis of the atomic
coordinates and numbered according to the residue assignments
deposited under entry 2AP2 at the BNL-pdb. .sup.5Residue regions
are defined within the diabody fragment from analysis of the atomic
coordinates and numbered according to the residue assignments
deposited under entry 1LMK at the BNL-pdb.
[0100] In another embodiment, the resulting Fab fragment contains
an antigen binding domain, at the N-terminal proximal end of the
molecule. The Fab fragment also contains a joining element that is
a peptide epitope, inserted at a position in the Fab fragment
replacing a defined .beta.-turn motif, or linked directly to the
distal C-terminal end of the Fab fragment. Thus the peptide epitope
fused to the Fab fragment serves as a highly specific joining
element that can serve as an attachment point, through the
recognition and binding of a cognate immunoconjugated functional
moiety.
[0101] Antibody Structural Elements
[0102] Antibodies are multivalent molecules made up of polypeptide
chains including light (L) chains of approximately 220 amino acids
and heavy (H) chains of 450-575 amino acids. The average molecular
weight for an intact IgG molecule is in the range 152-196 kD.
Structural studies performed on antibodies have revealed that both
the light and heavy chains contain a characteristic domain termed
the "immunoglobulin fold." The immunoglobulin fold is defined as a
barrel-shaped sandwich consisting of two layered anti-parallel
i-sheets linked together by a disulfide bond. The predominant
secondary structure in an antibody is an anti-parallel .beta.-sheet
with short stretches of .alpha.-helix. (For review, see Padlan,
1994, Anatomy of the antibody molecule, Mol. Immunol. 31(3):
169-217; Padlan, 1996, X-ray crystallography of antibodies, Adv.
Protein Chem. 49: 57-133; and references cited therein.)
[0103] The light chains contain two immunoglobulin domains, one at
the N-terminal portion, which varies from antibody to antibody
(V.sub.L), and the other at the C-terminal portion, which is
relatively constant (C.sub.L). The heavy chains contain four or
five immunoglobulin domains, depending upon the class of
immunoglobulin. The N-terminal domain varies (V.sub.H) and the
other distal domains remain constant (C.sub.H1, C.sub.H2, C.sub.H3,
and, in certain cases C.sub.H4). The units of the light and heavy
chains associate through disulfide bonds as well as other
non-covalent interactions to form the characteristic Y-shaped dimer
composed of two light chains and two heavy chains. The antibody
fragment containing the V.sub.L chain and the V.sub.H chain is
termed the Fv fragment. The portion containing the entire light
chain, as well as the variable portion and first constant domain
(C.sub.H1) of the heavy chain, is termed the Fab fragment.
Interactions of the variable domains with the constant domains in
Fab are not very strong, lending a degree of flexibility and
positional variability to the overall structure of the molecule.
There can be a large variation (from 127-176.degree.) in the angle
between the Fab variable domain and the Fab constant domain. This
angle is known as the Fab "elbow" or "bend" (Padlan, 1994, Anatomy
of the antibody molecule. Mol. Immunol. 31(3): 169-217).
[0104] The N-terminal regions of the two Fab arms bind antigen
(Mian et al., 1991, Structure, function and properties of antibody
binding sites, J. Mol. Biol. 217(1): 133-51; Wilson et al., 1994,
Structure of anti-peptide antibody complexes, Res. Immunol. 145(1):
73-8; Wilson et al., 1994, Antibody-antigen interactions: new
structures and new conformational changes, Curr. Opin. Struct.
Biol. 4(6): 857-67). The Fab arms, in turn, are connected by a
flexible polypeptide to the third fragment, termed the Fc fragment,
which is responsible for triggering effector functions that
eliminate the antigen as well as dimerize the antigen binding
sites.
[0105] The Fc portion of the IgG antibody molecule is made up of
the two constant domains C.sub.H2 and C.sub.H3. The polypeptide
segment connecting the Fab and Fc fragments is defined as the hinge
and has variable length and flexibility depending upon the antibody
class and isotype. This flexible hinge region provides a natural
demarcation between the Fc and Fab fragments of the antibody. The
hinge and the Fab elbow or bend contained in an intact IgG molecule
allow for significant flexibility between the two antigen binding
sites and thus permit numerous cross-linking geometries (FIGS. 5
and 6).
[0106] The proteins making up native and recombinant antibody
fragments are candidates for the structural elements of
nanostructures assembled by staged assembly. Antibodies used in the
staged assembly methods of the invention include, but are not
limited to, IgG monoclonal, humanized or chimeric antibodies.
Binding derivatives or binding fragments of antibodies used in the
staged assembly methods of the invention also include, but are not
limited to, single chain antibodies (scFv) including monomeric
((scFv) fragments), dimeric ((scFv).sub.2 or diabodies), trimeric
((scFv).sub.3 or triabodies) and tetrameric ((scFv).sub.4 or
tetrabodies) single chain antibodies; Fab fragments; F(ab').sub.2
fragments; and fragments produced by a Fab expression library (Huse
et al., 1989, Generation of a large combinatorial library of the
immunoglobulin repertoire in phage lambda, Science, 246,
1275-81).
[0107] Antibody Production
[0108] General methods of antibody production and use are commonly
known in the art. These methods may be used for producing
structural and joining elements for use in the staged assembly
methods and assembly units of the invention (see, e.g., Harlow and
Lane, 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.; incorporated herein by
reference in its entirety).
[0109] A molecular clone of an antibody to an antigen of interest
can be prepared by techniques well-known in the art. Recombinant
DNA methodology may be used to construct nucleic acid sequences
that encode a monoclonal antibody molecule, or antigen binding
region thereof (see, e.g., Sambrook et al., 2001, Molecular
Cloning, A Laboratory Manual, Third Edition, Chapters 1, 2, 3, 5,
6, 8, 9, 10, 13, 14, 15 and 18, Cold Spring Harbor Laboratory
Press, N.Y.; Ausubel et al., 1989, Current Protocols in Molecular
Biology, Chapters 1, 2, 3, 5, 6, 8, 10, 11, 12, 15, 16, 19, 20 and
24, Green Publishing Associates and Wiley Interscience, N.Y.;
Current Protocols in Immunology, Chapters 2, 8, 9, 10, 17 and 18,
John Wiley & Sons, 2001, Editors John E. Coligan, Ada M.
Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober,
Series Editor: Richard Coico).
[0110] Antibodies can be expressed in bacteria either
intracellularly or extracellularly by secretion into the bacterial
periplasm (Tomlinson and Holliger, 2000, Methods for generating
multivalent and bispecific antibody fragments, Methods Enzymol.
326: 461-79). Intracellular expression of recombinant antibodies,
however, frequently leads to the formation of insoluble aggregates
of the protein, which are referred to as inclusion bodies,
presumably due to the non-reducing environment of the bacterial
cytoplasm, which inhibits disulfide bond formation between antibody
domains. It is possible to refold the antibodies into functional
proteins through solubilization of the inclusion bodies with strong
denaturants followed by exposure to renaturing conditions, by
methods commonly known in the art.
[0111] In order to circumvent the need for renaturation, a coding
sequence for a bacterially-derived periplasmic signal sequence can
be spliced at the N-terminal portion of the gene encoding the
antibody to direct the recombinant protein to the bacterial
periplasm. The oxidizing environment of the periplasmic space
favors proper folding of the antibody domains, including disulfide
bond formation. The success of these methods in producing good
yields of functional antibody can depend upon the antibody type,
derivation and method of overproduction (see Ward, 1992, Antibody
engineering: the use of Escherichia coli as an expression host,
FASEB J. 6(7): 2422-27; Ward, 1993, Antibody engineering using
Escherichia coli as host, Adv. Pharmacol. 24: 1-20; Zhu et al.,
1996, High level secretion of a humanized bispecific diabody from
Escherichia coli, Biotechnology (NY) 14(2): 192-96; Sheets et al.,
1998, Efficient construction of a large nonimmune phage antibody
library: the production of high-affinity human single-chain
antibodies to protein antigens, Proc. Natl. Acad. Sci. USA 95(11):
6157-62; Tomlinson et al., 2000, Methods for generating multivalent
and bispecific antibody fragments, Methods Enzymol. 326:
461-79).
[0112] Antibody molecules may be purified by techniques well-known
in the art, e.g., immunoabsorption or immunoaffinity
chromatography, chromatographic methods such as HPLC (high
performance liquid chromatography), or a combination thereof.
[0113] Structural Elements Comprising Monoclonal Antibodies
[0114] Monoclonal antibodies (mAbs), or binding derivatives or
binding fragments thereof, may be used as structural elements
according to the methods of the invention. mAbs are homogeneous
populations of antibodies directed against a particular antigen. A
mAb to an antigen of interest can be prepared by using any
technique known in the art that provides for the production of
antibody molecules. These include, e.g., the hybridoma technique
originally described by Kohler and Milstein (1975, Continuous
cultures of fused cells secreting antibody of predefined
specificity, Nature 256: 495-97; Voet and Voet, 1990, Biochemistry,
John Wiley and Sons, Inc., Chapter 34), the human B cell hybridoma
technique (Kozbor et al., 1983, Immunology Today 4: 72-79; Kozbor
et al., U.S. Pat. No. 4,693,975, entitled "Human hybridroma [sic]
fusion partner for production of human monoclonal antibodies,"
issued Sep. 15, 1987), and the EBV-hybridoma technique (Cole et
al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,
Inc., pp. 77-96; Roder et al., 1986, The EBV-hybridoma technique,
Methods Enzymol.121: 140-67). Such antibodies may be of any
immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any
subclass thereof. The mAbs that may be used in the methods of the
invention may be synthesized by any technique commonly known in the
art. For example, human monoclonal antibodies may be made by any of
numerous techniques known in the art (e.g., Teng et al., 1983,
Construction and testing of mouse--human heteromyelomas for human
monoclonal antibody production, Proc. Natl. Acad. Sci. USA. 80:
7308-12; Cole et al., 1984, Human monoclonal antibodies, Mol. Cell.
Biochem. 62(2): 109-20; Olsson et al., 1982, Immunochemical
Techniques, Meth. Enzymol. 92: 3-16).
[0115] By contrast, polyclonal antibodies cannot be used as
components in the present invention. Polyclonal antibodies
represent a population of antibodies in which many molecules of
different precise specificity exists. Although they may all bind to
a particular antigen, they will bind different parts of the antigen
with different geometries, a property that is inconsistent with the
precise assembly of a nanostructure.
[0116] Structural Elements Comprising Multispecific Antibodies
[0117] Multispecific antibodies, or binding derivatives or binding
fragments thereof, may be used as structural elements for use in
the staged assembly methods of the invention. "Specific" or
"specificity," as used herein, refers to the ability of an antibody
to bind a defined epitope to one distinct antigen-recognition site.
Bispecific antibodies, therefore, comprise two distinct antigen
recognition sites, each capable of binding a different antigen.
Multispecific antibodies have the ability to bind more than two
different epitopes, each through the action of a distinct joining
element, i.e., an antigen-recognition site.
[0118] In certain embodiments, homogeneous bispecific or
multispecific mAbs can be created for use as structural elements,
via immortilization of lymphocyte clones, created by fusing myeloma
cells with lymphocytes raised against an antigen of interest as
described above generally for the production of monoclonal
antibodies. By such methods, multispecific mAbs can be produced in
virtually unlimited quantities. Using methods well-known in the
art, multispecific mAbs may be created that specifically target and
bind a selected biological substance (see, e.g., Colcher et al.,
1999, Single-chain antibodies in pancreatic cancer, Ann. NY Acad.
Sci. 880: 263-80; Hudson, 1999, Recombinant antibody constructs in
cancer therapy, Curr. Opin. Immunol. 11 (5): 548-57; Kipriyanov et
al., 1999, Bispecific tandem diabody for tumor therapy with
improved antigen binding and pharmacokinetics, J. Mol. Biol.
293(1): 41-56; Segal et al., 1999, Bispecific antibodies in cancer
therapy, Curr. Opin. Immunol. 11(5): 558-62; Trail et al., 1999,
Monoclonal antibody drug conjugates in the treatment of cancer,
Curr. Opin. Immunol. 11(5): 584-88; Hudson, 2000, Recombinant
antibodies: a novel approach to cancer diagnosis and therapy,
Expert Opin. Investig. Drugs 9(6): 1231-42).
[0119] In certain embodiments, a multispecific mAb for use as a
structural element according to the methods of the invention may be
a bispecific and/or bivalent mAb. A bispecific antibody has the
ability to bind two different epitopes, each contained on a
distinct antigen-recognition site. A bivalent antibody has the
ability to bind to two different epitopes.
[0120] Bispecific antibodies may be created using methods
well-known in the art (see, e.g., Weiner et al., 1995, Bispecific
monoclonal antibody therapy of B-cell malignancy, Leuk. Lymphoma
16(3-4): 199-207; Helfrich et al., 1998, Construction and
characterization of a bispecific diabody for retargeting T cells to
human carcinomas, Int. J. Cancer 76(2): 232-39; Arndt et al., 1999,
A bispecific diabody that mediates natural killer cell cytotoxicity
against xenotransplanted human Hodgkin's tumors, Blood 94(8):
2562-8; Kipriyanov et al., 1999, Bispecific tandem diabody for
tumor therapy with improved antigen binding and pharmacokinetics,
J. Mol. Biol. 293(1): 41-56; Sundarapandiyan et al., 2001,
Bispecific antibody-mediated destruction of Hodgkin's lymphoma
cells, J. Immunol. Methods 248(1-2): 113-23).
[0121] Technologies for the production of multivalent and
multispecific antibodies are well known in the art (see, e.g.,
Pluckthun et al., 1997, New protein engineering approaches to
multivalent and bispecific antibody fragments, Immunotechnology
3(2): 83-105; Santos et al., 1998, Development of more efficacious
antibodies for medical therapy and diagnosis, Prog. Nucleic Acid
Res. Mol. Biol. 60: 169-94; Alt et al., 1999, Novel tetravalent and
bispecific IgG-like antibody molecules combining single-chain
diabodies with the immunoglobulin gamma1 Fc or CH3 region, FEBS
Lett. 454(1-2): 90-94; Hudson et a/., 1999, High avidity scFv
multimers; diabodies and triabodies, J. Immunol. Methods 231(1-2):
177-89; Tomlinson et al., 2000, Methods for generating multivalent
and bispecific antibody fragments, Methods Enzymol. 326: 461-79;
Todorovska et al., 2001, Design and application of diabodies,
triabodies and tetrabodies for cancer targeting. J. Immunol.
Methods 248(1-2): 47-66). For example, genes encoding antibodies of
known specificity may be rescued from hybridoma cell lines and can
provide the starting material for cloning the rearranged V.sub.L
and V.sub.H genes thorough employment of recombinant DNA
technologies (Ward et al., 1989, Binding activities of a repertoire
of single immunoglobulin variable domains secreted from Escherichia
coli, Nature 341(6242): 544-46; Sheets et al., 1998, Efficient
construction of a large nonimmune phage antibody library: the
production of high-affinity human single-chain antibodies to
protein antigens, Proc. Natl. Acad. Sci. USA 95(11): 6157-62).
Universal DNA primers may be designed to anneal to the target
V-domain genes and amplified through employment of the polymerase
chain reaction. Through design of restriction sites within these
primers, the resulting amplified DNA products can be cloned
directly for expression in a range of different hosts including
bacteria, yeast, plant and insect cells (Tomlinson et al., 2000,
Methods for generating multivalent and bispecific antibody
fragments, Methods Enzymol. 326: 461-79). These host cells, rather
than hybridoma cell lines, can be used, for the production of
recombinant engineered antibodies for use in the methods of the
invention.
[0122] In certain embodiments of the invention, a structural
element comprises a diabody fragment. A diabody has two CDRs, and
is capable of making two highly specific, non-covalent
interactions. A diabody, or a binding derivative or binding
fragment thereof, may be incorporated into a nanostructure in such
a way that only one of the two CDRs is used. In certain
embodiments, the CDRs themselves serve as joining elements, and the
body of the diabody between the two CDRs serves as a structural
element.
[0123] Methods well known in the art are used for the expression,
purification and characterization of diabody fragments from E. coli
(Poljak, 1994, An idiotope--anti-idiotope complex and the
structural basis of molecular mimicking, Proc. Natl. Acad. Sci. USA
91(5): 1599-600; Zhu et al., 1996, High level secretion of a
humanized bispecific diabody from Escherichia coli, Biotechnology
(NY) 14(2): 192-96; Todorovska et al., 2001, Design and application
of diabodies, triabodies and tetrabodies for cancer targeting, J.
Immunol. Methods 248(1-2): 47-66). Examples of a structural element
comprising a diabody fragment are illustrated in FIG. 4. The
diabody expression cassettes represented in FIG. 4 are designed so
that the pelB signal sequence spliced to the N-terminus of the
V.sub.H domains genes coding the diabody fragments are targeted and
secreted into the E. coli periplasmic space, where the oxidative
environment allows proper folding of the diabody. After induction,
the overexpressed diabodies fragments are harvested from the E.
coli periplasm according to established protocols well-known in the
art.
[0124] In a preferred embodiment of the invention (FIG. 4),
diabodies are engineered to add a hexahistidine tag (His6) at the
C-terminus of the V.sub.L domains to facilitate purification using
an immobilized metal affinity chromatography resin (Scopes, 1994,
Protein Purification, Principles and Practice, Third Edition,
Springer-Verlag, London, pp. 183-85; Scopes, 1994 Protein
Purification: Principles and practice (Springer Advanced texts in
Chemistry), Third ed., London). Protein overexpression of diabody
assembly unit-1 (FIG. 4A), for example, will contain a mixture of
species including; 2 (V.sub.HA.times.V.sub.LBHiS.sub- .6),
2(V.sub.HB.times.V.sub.LA), and (V.sub.HB.times.V.sub.LA,
V.sub.HA.times.V.sub.LBHiS.sub.6). The number of His.sub.6 tags
determines the concentration of imidazole (20-250 mM gradient) at
which each protein unit contained in the mixture will elute. Those
with no hexahistidine tags will exhibit little or no affinity
towards the column resin. Those with one hexahistidine tag will
generally elute between 20-40 mM imidazole (bispecific diabody) and
those with two hexahistidine tags will generally elute between 50
and 100 mM imidazole. Elution peaks may be detected by UV
absorbance and verified with SDS-PAGE, native-PAGE or ELISA assay.
Even though the purification procedure described above guards
against the isolation of unwanted non-bispecific diabody
byproducts, methods are employed to ensure that the isolated
diabody of interest has functional bispecificity as disclosed
hereinbelow.
[0125] FIG. 4A depicts an A.times.B diabody in which the V.sub.H
and V.sub.L domains of A define a lysozyme isotopic antibody (D1.3)
and in which the V.sub.H and V.sub.L domains of B define a virus
neutralizing idiotopic antibody (730.1.4). In order to facilitate
purification of the desired diabody product, the gene encoding
V.sub.HA and V.sub.LB includes a hexahistidine tag, whereas the
gene encoding V.sub.HB and V.sub.LA does not. FIG. 4B depicts a
B'.times.A' diabody in which the V.sub.H and V.sub.L domains of B'
define a virus neutralizing idiotopic antibody (409.5.3) and in
which the V.sub.H, and V.sub.L domains of A' define a lysozyme
isotopic antibody (E5.2). In order to facilitate purification of
the desired diabody product, the gene encoding V.sub.HB' and
V.sub.LA' includes a hexahistidine tag, whereas the gene encoding
V.sub.HA' and V.sub.LB' does not.
[0126] In certain embodiments, sandwich ELISA or BIAcore protocols
may be implemented to determine simultaneous and dual occupancy of
both antigen-binding sites (bispecificity), as well as equilibrium
constants (Abraham et al., 1996, Determination of binding constants
of diabodies directed against prostate-specific antigen using
electrochemiluminescence- -based immunoassays, J. Mol. Recognit.
9(5-6): 456-61; McGuinness et al., 1996, Phage diabody repertoires
for selection of large numbers of bispecific antibody fragments,
Nat. Biotechnol. 14(9): 1149-54; McCall et al., 2001, Increasing
the affinity for tumor antigen enhances bispecific antibody
cytotoxicity, J. Immunol. 166(10): 6112-17). In a specific
embodiment in which an idiotype/anti-idiotype binding constant is
determined using the BIAcore technique, one of the antibodies is
dissolved in a liquid phase and the other is coupled to the solid
phase. Implementation of this technique permits the determination
of the association and dissociation rates (k.sub.on, and k.sub.off
respectively) for determination of the dissociation constant (Kd)
(Goldbaum et al., 1997, Characterization of anti-anti-idiotypic
antibodies that bind antigen and an anti-idiotype, Proc. Natl.
Acad. Sci. USA 94(16): 8697-701). Other protocols that do not
require recombinant antigens, but that can detect bispecificity may
also be employed, and include the rosetting assay as described by
Holliger et al. (1997, Retargeting serum immunoglobulin with
bispecific diabodies, Nat. Biotechnol. 15(7): 632-36).
[0127] In a specific embodiment, a diabody may comprise one or more
sites for the insertion of a joining element, a structural element
or a functional element. Table 4 shows peptide regions contained in
diabody units that may be used for the insertion of joining,
structural or functional elements. A peptide region is a portion of
a protein of interest, e.g., of an antibody or a binding derivative
or binding fragment thereof. A peptide region is preferably exposed
on the surface of the protein of interest, and is amenable to being
re-engineered through the insertion of additional peptides or the
alteration of its sequence or both. Table 4 summarizes the amino
acids identified as .beta.-turns located on the surface of a
diabody with V.sub.H-V.sub.L variable domain linkage (pdb entry
1LMK). Residue regions are defined within the diabody fragment from
analysis of the atomic coordinates and numbered according to the
residue assignments deposited under entry 1LMK pdb. Chain
assignments are labeled in accord with the corresponding deposited
pdb coordinates.
4TABLE 4 Identified Peptide Regions Contained in Diabody Structural
Elements for the Insertion of Joining, Structural or Functional
Elements Domain Secondary Structure Residue (Chain) V.sub.H
.beta.-turn Residues 13-16, 39-44, 62-66, 73-77 (A and C chains)
V.sub.L C-terminal .alpha.-C Residue 312 (A and C chains) V.sub.H
C-terminal .alpha.-C Residue 1 (A and C chains)
[0128] In certain embodiments, binding sites may be added as
joining elements to a diabody to make possible structural branches,
forks, T-junctions, or multidimensional architectural binding
sites, in addition to the two joining elements formed by the
oppositely directed CDRs. Alteration of the sequence of surface
loops in proteins appears to have little impact on the overall
folding of a protein, and it is frequently possible to make
insertion mutants at the sites of .beta.-turns. The surface loops
are the places where sequences can be added to the protein with the
lowest probability of disrupting the protein structure.
[0129] Specific sites within the diabody unit have been precisely
defined for insertions. For example, in certain embodiments,
joining elements mays be spliced internal to, or replacing the
.beta.-turn residues as disclosed herein in Table 4. Since the
general three-dimensional structure of diabodies is known, and
since it is possible to homology-model the three-dimensional
structure of diabodies of similar sequence (Guex and Peitsch, 1997,
SWISS-MODEL and the Swiss-PdbViewer: An environment for comparative
protein modelling, Electrophoresis 18: 2714-23; Guex and Peitsch,
1999, Molecular modelling of proteins, Immunology News 6: 132-34;
Guex et al., 1999, Protein modelling for all, TIBS 24: 364-67, the
.beta.-turns located on the surface of a diabody of similar amino
acid sequence to a diabody of known structure are readily
identified by a sequence comparison (using, e.g., BLAST, Altschul
et al., 1997, Gapped BLAST and PSI-BLAST: a new generation of
protein database search programs, Nucleic Acids Res. 25:
3389-3402), followed by a visual investigation of the x-ray
coordinates of the protein of similar sequence.
[0130] In one embodiment, a visual investigation of the
three-dimensional structure of a diabody is performed with the
molecular visualization package QUANTA (Accelrys Inc., San Diego,
Calif.) run on a Silicon Graphics Workstation. The coordinates
defining the three-dimensional positions of the atoms of a diabody
molecule are included in the PDB entry 1LMK. Upon such an analysis,
it is apparent that there are surface loops that include residues
shown in Table 4, which represent sites with high potential for
accepting the insertion of a peptide such as the HIV-1 V3 loop
antigen. All amino acids included in surface loops of this diabody
molecule can be determined from this information, and the relative
spatial locations of these surface loops has also been determined.
The information provided by the three-dimensional structure of the
immunoglobulin being engineered (whether derived directly from
X-ray crystallography, or from homology modeling based on a
homologous structure) allows the identification of all the amino
acids in the protein of interest that correspond to amino acids
that constitute surface loops.
[0131] In a specific embodiment, DNA encoding a peptide epitope
derived from the H-ras protein is inserted into a diabody assembly
unit coding sequence at a site defined by visual investigation of
the three-dimensional atomic coordinates as determined by x-ray
crystallography. The H-ras epitope is flanked by four glycines on
either side, to provide flexibility and accessibility for cognate
antibody binding.
[0132] Once the diabody assembly unit/ras peptide protein fusion
(represented as B.sup.ras.times.A) has been expressed and purified,
it is characterized for retention of diabody valency and function
as well as epitope recognition by the appropriate antibody by
methods such as ELISA or BIAcore analysis.
[0133] Functional elements, such as enzymes, toxins, and antigenic
peptides, have already been successfully spliced to the termini of
scFv fragments resulting in various multifunctional antibodies
(Chaudhary et al., 1989, A recombinant immunotoxin consisting of
two antibody variable domains fused to Pseudomonas exotoxin, Nature
339(6223): 394-97; Suzuki et al., 1997, Construction, bacterial
expression, and characterization of hapten-specific single-chain Fv
and alkaline phosphatase fusion protein, J. Biochem. (Tokyo)
122(2): 322-29; Williams et al., 2001, Numerical selection of
optimal tumor imaging agents with application to engineered
antibodies, Cancer Biother. Radiopharm. 16(1): 25-35). Functional
elements that are made up of proteins or peptides can be fused
directly into the proteinaceous portion of an assembly unit using
the methods of molecular biology followed by expression of the
proteins in appropriate host.
[0134] Structural Elements Comprising Fab or F(ab').sub.2 Antibody
Fragments
[0135] In certain embodiments of the invention, a structural
element for the staged assembly of a nanostructure comprises an
antibody fragment. Such a fragment includes, but is not limited to,
an Fab fragment, or an F(ab').sub.2 fragment, which can be produced
by pepsin digestion of an IgG antibody molecule, thereby releasing
the Fc portion. Pepsin digestion can be followed by reducing the
disulfide bridges between the resulting F(ab').sub.2 fragments
thereby generating single Fab fragments.
[0136] Fab fragments are elongated dirigible shaped molecules that
contain a monovalent and monospecific CDR at the N-terminal end of
the molecule. In certain embodiments, an assembly unit is
engineered from a Fab fragment by inserting a peptide epitope at
the C terminal portion of the Fab fragment. Consequently, a peptide
fused to the C-terminus of the Fab fragment may act as a target for
another engineered Fab, to provide a highly specific and tight
interaction between adjacent Fabs in a nanostructure constructed by
staged assembly. The size, shape and structure of the Fab fragment
(FIG. 6) make it preferred for use as a structural element because
it also comprises, by virtue of its structure, a naturally
occurring joining element. Electron micrographic and X-ray
structural studies have revealed that the proximal portion of the
Fab fragment is often linearly opposed to the distal portion
(Fischmann et al., 1991, Crystallographic refinement of the
three-dimensional structure of the FabD1.3-lysozyme complex at 2.5
.ANG. resolution, J. Bio. Chem 266: 12915-20; Ban et al., 1994,
Crystal structure of an idiotype-anti-idiotype Fab complex, Proc.
Natl. Acad. Sci. U.S.A. 91(5): 1604-8; Padlan, 1996, X-ray
crystallography of antibodies, Adv. Protein Chem. 49: 57-133;
Harris, Skaletsky et al., 1998). The flexible elbow bend, which is
located in the middle of the fragment, allows for alternative
geometries (Roux et al., 1997, Flexibility of human IgG subclasses,
J. Immunol. 159(7): 3372-82; Roux et al., 1998, Comparisons of the
ability of human IgG3 hinge mutants, IgM, IgE, and IgA2, to form
small immune complexes: a role for flexibility and geometry, J.
Immunol. 161(8): 4083-90). Alternatively, Fab expression libraries
may be constructed (Huse et al., 1989, Generation of a large
combinatorial library of the immunoglobulin repertoire in phage
lambda, Science, 246, 1275-81) to allow rapid and easy
identification of monoclonal Fab fragments with the desired
specificity.
[0137] Structural Elements Comprising Single-Chain Antibody
Fragments (scFvs)
[0138] According to the methods of the invention, staged assembly
of nanostructures can employ, in certain embodiments, structural
elements comprising single-chain scFv fragments. An scFv antibody
is composed of a fusion peptide that links the carboxyl terminus of
the Fv variable heavy chain (V.sub.H) to the amino terminus of the
Fv variable light chain (V.sub.L) or vice versa (Freund et al.,
1994, Structural and dynamic properties of the Fv fragment and the
single-chain Fv fragment of an antibody in solution investigated by
heteronuclear three-dimensional NMR spectroscopy, Biochemistry
33(11): 3296-303; Hudson et al., 1999, High avidity scFv multimers;
diabodies and triabodies, J. Immunol. Methods 231(1-2): 177-89; Le
Gall et al., 1999, Di-, tri- and tetrameric single chain Fv
antibody fragments against human CD19: effect of valency on cell
binding, FEBS Lett 453(1-2): 164-68; Worn et al., 2001, Stability
engineering of antibody single-chain Fv fragments, J. Mol. Biol.
305(5): 989-1010).
[0139] Single-chain antibodies may also be used as structural
elements for use in the staged assembly methods of the invention.
Single-chain antibodies may be produced by the methods of, e.g.,
Ladner; (U.S. Pat. No. 4,946,778, entitled "Single polypeptide
chain binding molecules," issued Aug. 7, 1990); Bird (1988,
Single-Chain Antigen-Binding Proteins, Science 242(4877): 423-26);
Huston et al. (1988, Protein engineering of antibody binding sites:
recovery of specific activity in an anti-digoxin single-chain Fv
analogue produced in Escherichia coli, Proc. Natl. Acad. Sci. USA
85: 5879-83), or Ward et al., (1989, Binding activities of a
repertoire of single immunoglobulin variable domains secreted from
Escherichia coli, Nature 334: 544-46).
[0140] An scFv fragment is a substructure of a Fab fragment that
can be visualized as a Fab fragment, cut in half at the elbow-bend,
missing the terminal constant light and heavy chain domains Freund
et al., 1994, Structural and dynamic properties of the Fv fragment
and the single-chain Fv fragment of an antibody in solution
investigated by heteronuclear three-dimensional NMR spectroscopy,
Biochemistry 33(11): 3296-303; Malby et al., 1998,
Three-dimensional structures of single-chain Fv-neuraminidase
complexes, J. Mol. Biol. 279(4): 901-10) (FIG. 2). Rather than
being elongated and dirigible shaped, as in Fab fragments, scFv are
smaller and more globular shaped. While approximately half the size
of a Fab fragment, a scFv fragment still contains a functional
monovalent/monospecific CDR at the N-terminal portion of the
molecule. The scFv represents the minimal antigen binding motif
that can be expressed in E. coli.
[0141] In general, scFv fragments are monovalent, maintaining
tertiary and quaternary structures similar to that found in the Fv
portion of an intact antibody (FIGS. 5 and 2) (Boulot et al., 1990,
Crystallization and preliminary X-ray diffraction study of the
bacterially expressed Fv from the monoclonal anti-lysozyme antibody
D1.3 and of its complex with the antigen, lysozyme, J. Mol. Biol.
213(4): 617-19; Braden et al., 1996, Crystal structure of an Fv-Fv
idiotope-anti-idiotope complex at 1.9 .ANG. resolution, J. Mol.
Biol. 264(1): 137-51; Fuchs et al., 1997, Primary structure and
functional scFv antibody expression of an antibody against the
human protooncogene c-myc, Hybridoma 16(3): 227-33; Hoedemaeker et
al., 1997, A single chain Fv fragment of P-glycoprotein-specific
monoclonal antibody C219. Design, expression, and crystal structure
at 2.4 .ANG. resolution, J. Biol. Chem. 272(47): 29784-89; Malby et
al., 1998, Three-dimensional structures of single-chain
Fv-neuraminidase complexes, J. Mol. Biol. 279(4): 901-10). A
Gly/Ser peptide linker that is, optimally, 15 amino acids in
length, can be used to join the two variable fragments and help
maintain favorable interactions between the V.sub.H and V.sub.L
domains (Perisic et al. 1994, Crystal structure of a diabody, a
bivalent antibody fragment, Structure 2(12): 1217-26; Takemura et
al, 2000, Construction of a diabody (small recombinant bispecific
antibody) using a refolding system, Protein Eng. 13(8): 583-88;
Worn et al., 2001, Stability engineering of antibody single-chain
Fv fragments, J. Mol. Biol. 305(5): 989-1010). These Gly/Ser
linkers can be used to provide flexibility and protease resistance.
Furthermore, scFv antibody fragments have similar function, in
terms of antigen recognition and binding, as that of intact
antibodies.
[0142] The smaller size of the scFv fragment, as well as the
relative positioning of the CDR, make it well-suited as a protein
component to be incorporated into assembly units of the present
invention for fabrication of nanostructures. One advantage of scFv
over Fab fragments is that the technology for engineering and
producing scFv's is more advanced (see, e.g., Ward, 1993, Antibody
engineering using Escherichia coli as host, Adv. Pharmacol. 24:
1-20; Luo et al., 1996, Construction and expression of
bi-functional proteins of single-chain Fv with effector domains, J.
Biochem. (Tokyo) 120(2): 229-32; Wu et al., 2000, Designer genes:
recombinant antibody fragments for biological imaging, Q. J. Nucl.
Med. 44(3): 268-83; Worn et al., 2001, Stability engineering of
antibody single-chain Fv fragments, J. Mol. Biol. 305(5):
989-1010). Using these art-known methods, specific CDRs may be
created, and functional elements may be added to scFv's for use as
protein components to be incorporated into assembly units useful in
for staged assembly of nanostructures.
[0143] In another embodiment, a similar strategy is used to
incorporate additional intermolecular binding sites on the scFv as
was described above for Fab fragments. The C-terminal distal
portion or .beta.-turn regions can be replaced by defined peptide
epitopes such as, but not limited to those provided in Table 2,
below. These peptide epitopes can replace defined .beta.-turn
motifs or be directly linked to the C-terminal amino acid of the
V.sub.H or V.sub.L heavy chain (depending upon the order of the
linked heavy and light variable domains) (Table 3), by manipulation
of the appropriate encoding DNA sequences using recombinant DNA
procedures well known in the art. The resulting scFv fragment will
contain an antigen binding recognition site on one portion of the
scFv fragment and a joining element that is a peptide epitope,
either replacing the defined .beta.-turn motifs, or linked at the
C-terminal portion of the scFv fragment. Thus the fused peptide
epitope will serve as a highly specific joining element in the
formation of a joining pair between adjacent assembly units
comprising scFv in a staged assembly.
[0144] Structural Elements Comprising Bispecific IgG, Chimeric IgG
or Bispecific Heterodimeric F(ab').sub.2 Antibodies
[0145] In certain embodiments of the invention, a structural
element comprises an antibody fragment such as a bispecific IgG
fragment, chimeric IgG fragment or a bispecific heterodimeric
F(ab').sub.2 antibody fragment. Whereas naturally occurring IgG
molecules are bivalent by design, but monospecific because their
CDRs are identical, IgG molecules, such as those created by
hybridoma technology, can be produced that are either bivalent or
bispecific, using the methods of, e.g., Suresh et al. (1986,
Bispecific monoclonal antibodies from hybrid hybridomas, Methods
Enzymol. 121: 210-28); Holliger et al. (1993, Engineering
bispecific antibodies, Curr. Opin. Biotechnol. 4(4): 446-49);
Hayden et al. (1997, Antibody engineering, Curr. Opin. Immunol.
9(2): 201-12); Carter (2001, Bispecific human IgG by design, J.
Immunol. Methods 248(1-2): 7-15).
[0146] Bispecific IgGs may be created by any method known in the
art, e.g., by chemical coupling methodologies or through the
development of hybrid hybridoma cell lines (also referred to as
hybrid hybridoma technology) (Milstein et al., 1983, Hybrid
hybridomas and their use in immunohistochemistry, Nature 305(5934):
537-40) (FIG. 7).
[0147] Another approach used to obtain bispecific antibodies
comprises exposing IgG to limited proteolytic digestion, where the
two identical Fab fragments are released from the Fc fragment upon
cleavage of the hinge polypeptide (FIG. 8). These single monovalent
Fab fragments can be used alone, or chemically linked together (at
the hinge cysteines) with a Fab fragment of separate origin to form
a bispecific heterodimeric F(ab').sub.2. Chemically linked
bispecific F(ab').sub.2 fragments have been studied and evaluated
in several small-scale clinical trials (Hudson, 1999, Recombinant
antibody constructs in cancer therapy, Curr. Opin. Immunol. 11(5):
548-57; Segal et al., 1999, Bispecific antibodies in cancer
therapy, Curr. Opin. Immunol. 11(5): 558-62). Several other
rational design strategies have been developed in order to engineer
the Fc portion of heavy chains to promote the heterodimerization of
bispecific antibodies. These strategies can include, for example,
steric complementarity design mutations ("knobs-into-holes"
utilizing phage display technology) as well as the design of
additional inter-chain disulfide bonds and/or salt-bridge
interactions between the heavy chains of the Fc fragment (Carter
2001, Bispecific human IgG by design, J. Immunol. Methods 248(1-2):
7-15). The enhanced complementarity between heavy chains of a
desired bispecific antibody makes bispecific antibodies a preferred
source for structural elements for use in the staged assembly of
nanostructures as disclosed herein.
[0148] In one embodiment, bispecific antibodies are produced by
replacing the Fc dimer-forming motif with another dimerization
motif. In one non-limiting example, leucine zippers that can form
heterodimers, such those found in Fos and Jun proteins, are linked
to two different Fab portions of an IgG molecule by gene fusion.
When expressed individually in an appropriate cell line, the fusion
IgG's can be isolated as Fab-(zipper).sub.2 homodimers. Heterodimer
formation is then achieved by reduction of the disulfide bonds
within the hinge region of the homodimers to release the monomeric
subunits.
[0149] The resulting monomers are mixed together and placed under
oxidizing conditions, resulting in bispecific heterodimers
containing Fos-Jun paired leucine zipper motifs as the majority of
the end products. Variations of this technique can be used to
produce bispecific Fab and Fv fusion proteins (Kostelny et al,
1992, Formation of a bispecific antibody by the use of leucine
zippers, J. Immunol. 148(5): 1547-53; Tso et al., 1995, Preparation
of a bispecific F(ab').sub.2 targeted to the human IL-2 receptor,
J. Hematother. 4(5): 389-94; de Kruif et al., 1996, Leucine zipper,
dimerized bivalent and bispecific scFv antibodies from a
semi-synthetic antibody phage display library, J. Biol. Chem.
271(13): 7630-34). Additional multimerization motifs used to
promote bispecific dimer formation include, but are not limited to:
transcriptional factor p53 (Rheinnecker et al., 1996, Multivalent
antibody fragments with high functional affinity for a
tumor-associated carbohydrate antigen, J. Immunol. 157(7):
2989-97), streptavidin (Muller et al., 1998, A dimeric bispecific
mini-antibody combines two specificities with avidity, FEBS Lett.
432(1-2): 45-49), or helix-bundle motifs such as Rop (Pack et al.,
1993, Improved bivalent miniantibodies with identical avidity as
whole antibodies produced by high cell density fermentation of
Escherichia coli, Biotechnology 11: 1271-77; Dubel et al., 1995,
Bifunctional and multimeric complexes of streptavidin fused to
single chain antibodies (scFv), J. Immun. Methods 178: 201-09)
(FIG. 9). Such antibodies are useful in the present invention as a
source of a plurality of joining elements that are non-identical
and that do not interact with each other.
[0150] While the above-described methodologies permit the
production and isolation of bispecific antibodies, the methods also
result in the creation of mixtures of IgG products, in low yields
or combinations of both. Multivalent and multifunctional antibodies
of high quality, quantity and purity maybe created by recombinant
antibody technology ((see, e.g., Morrison et al., 1989, Genetically
engineered antibody molecules, Adv. Immunol. 44: 65-92; Shin et
al., 1993, Hybrid antibodies, Int. Rev. Immunol. 10(2-3): 177-86;
Sensel et al., 1997, Engineering novel antibody molecules, Chem.
Immunol. 65: 129-58; Hudson et al, 1998, Recombinant antibody
fragments, Curr. Opin. Biotechnol. 9(4): 395-402).
[0151] In other embodiments of the invention, human, humanized or
chimeric (e.g., human-mouse or human-other species) monoclonal
antibodies (mAbs), or binding derivatives or binding fragments
thereof, may be used as structural elements for use in the staged
assembly methods of the invention. Humanized antibodies are
antibody molecules from non-human species having one or more
complementarity determining regions (CDRs) from the non-human
species and a framework region from a human immunoglobulin
molecule. Humanized antibodies are also referred to as "chimeric
antibodies." Humanized or chimeric antibodies may be produced by
methods well known in the art (see, e.g., Queen, U.S. Pat. No.
5,585,089, entitled "Humanized immunoglobulins," issued Dec. 17,
1996, which is incorporated herein by reference in its
entirety).
[0152] Chimeric antibodies may be used as structural elements
according to the methods of the invention. A chimeric antibody is a
molecule in which different portions are derived from different
animal species, such as those having a variable region derived from
a murine mAb and a human immunoglobulin effector or constant
region. Techniques have been developed for the production of
chimeric antibodies (Morrison et al., 1984, Chimeric human antibody
molecules: mouse antigen-binding domains with human constant region
domains, Proc. Natl. Acad. Sci. USA 81: 6851-55; Neuberger et al.,
1984, Recombinant antibodies possessing novel effector functions,
Nature, 312, 604-08; Takeda et al., 1985, Construction of chimaeric
processed immunoglobulin genes containing mouse variable and human
constant region sequences, Nature 314: 452-54) by splicing the
genes from a mouse antibody molecule of appropriate antigen
specificity together with genes from a human antibody molecule of
appropriate biological or effector activity.
[0153] Structural Elements Comprising Diabodies or Multimeric scFv
Fragments
[0154] In certain embodiments of the invention, structural elements
comprise diabodies or multimeric scFv fragments. scFv fragments,
especially those with shortened peptide linkers, e.g. 3, 4 or 5
amino acid residues in length, form dimers ((scFv.sub.2) or
diabodies) rather than monomers in solution (Dolezal et al., 2000,
ScFv multimers of the anti-neuraminidase antibody NC10: shortening
of the linker in single-chain Fv fragment assembled in V(L) to V(H)
orientation drives the formation of dimers, trimers, tetramers and
higher molecular mass multimers, Protein Eng. 13(8): 565-74).
Interchain domain interactions, rather than intrachain domain
interactions, occur in order to form the stable dimeric diabody
fragments (Holliger et al., 1993, Diabodies: small bivalent and
bispecific antibody fragments, Proc. Natl. Acad. Sci. U.S.A.
90(14): 6444-48). A shortened peptide linker may prevent intrachain
domain pairing and thus allow formation of interchain interactions
that result in diabody fragment formation (Perisic et al. 1994,
Crystal structure of a diabody, a bivalent antibody fragment,
Structure 2(12): 1217-26).
[0155] In certain embodiments, diabodies can be used as the
structural elements for the staged assembly of one-, two- and
three-dimensional nanostructures. As used herein, the term
"diabody" refers to dimeric single-chain variable antibody
fragments (scFv). An scFv fragment, as described above, is composed
of a fusion peptide that links the carboxyl terminus of the Fv
variable heavy chain to the amino terminus of the Fv variable light
chain (V.sub.H-V.sub.L) or vice versa (i.e. V.sub.L-V.sub.H)
(Pluckthun et al., 1997, New protein engineering approaches to
multivalent and bispecific antibody fragments, Immunotechnology
3(2): 83-105; Hudson, 1998, Recombinant antibody fragments, Curr.
Opin. Biotechnol. 9(4): 395-402; Kipriyanov et al., 1999,
Generation of recombinant antibodies, Mol. Biotechnol.12(2):
173-201).
[0156] In certain embodiments, a diabody or multimeric fragment is
thermostable (see, e.g., Jermutus et al., 2001, Tailoring in vitro
evolution for protein affinity or stability, Proc. Natl. Acad. Sci.
USA 98(1): 75-80; Worn et al., 2001, Stability engineering of
antibody single-chain Fv fragments, J. Mol. Biol. 305(5):
989-1010). Thermostability is a useful characteristic for
structural elements utilized in the staged assembly of one- two-
and three-dimensional nanostructures.
[0157] Unlike a monovalent scFv fragment, a diabody is a bivalent
molecule containing "two bodies" that include two separate
antigen-binding sites in opposition to one another and related by
approximately 170.degree. about the pseudo-two-fold, axis of
symmetry (parallel to the interface) (Perisic et al., 1994, Crystal
structure of a diabody, a bivalent antibody fragment, Structure
2(12): 1217-26; Poljak, 1994, Production and structure of diabodies
Structure 2: 1121-23; Hudson et al., 1999, High avidity scFv
multimers; diabodies and triabodies, J. Immunol. Methods 231(1-2):
177-89) (FIG. 2).
[0158] A monospecific diabody contains two identical
antigen-binding sites, both with specificity for the same
ligand/hapten. A bispecific diabody contains two antigen-binding
sites, each specific for a different ligand/hapten; that is, a
bispecific diabody is derived from two different non-paired scFv
fragments. The first hybrid fragment contains the V.sub.H coding
region from a first F.sub.V antibody and a V.sub.L coding region
derived from a second F.sub.V antibody. The resulting
V.sub.H-V.sub.L hybrid fragment is joined together by a short
Gly/Ser linker. The second hybrid fragment contains the V.sub.L
coding region from the first F.sub.V antibody and the V.sub.H
coding region derived from the second F.sub.V antibody.
[0159] The use of bispecific links permits the creation of
bispecific antibody fragments that demonstrate bispecific affinity
towards each ligand (Poljak, 1994, An idiotope-anti-idiotope
complex and the structural basis of molecular mimicking, Proc.
Natl. Acad. Sci. U.S.A. 91(5): 1599-1600; Kipriyanov et al., 1998,
Bispecific CD3.times.CD19 diabody for T cell-mediated lysis of
malignant human B cells, Int. J. Cancer 77(5): 763-72; Arndt et
al., 1999, A bispecific diabody that mediates natural killer cell
cytotoxicity against xenotransplanted human Hodgkin's tumors, Blood
94(8): 2562-68; Takemura et al., 2000, Construction of a diabody
(small recombinant bispecific antibody) using a refolding system,
Protein Eng. 13(8): 583-88). Certain bispecific diabodies
demonstrate affinities towards ligands/haptens similar to that
demonstrated by whole IgG (Holliger et al., 1993, Engineering
bispecific antibodies, Curr. Opin. Biotechnol. 4(4): 446-49; Yagi
et al., 1994, Superantigen-like properties of an antibody
bispecific for MHC class II molecules and the V beta domain of the
T cell antigen receptor, J. Immunol. 152(8): 3833-41).
[0160] Diabodies exhibit several properties that make them
particularly attractive for use the in staged assembly methods of
the invention: (i) they are structures containing oppositely
directed antigen binding sites; (ii) the geometrical opposition of
the two antigen-binding sites optimizes the potential for building
linear nanostructures or linear extensions of nanostructures; (iii)
they have a well-defined size, shape, structure and stoichiometry;
(iv) they have structural rigidity and well-defined recognition and
binding properties; (v) binding motifs exhibiting specificity for a
very broad range of organic and inorganic moieties can be
identified and incorporated into a diabody structure (vi) their
X-ray structure has been solved (FIG. 2) and can serve as a
blueprint for identifying positions at which it is possible to add
functional groups or binding sites; (vii) diabodies form strong
intermolecular bonds to one another; (viii) the intermolecular
bonds are highly specific; (ix) the immunoglobulin fold provides a
structured protein core (structural element) and a stable spatial
relationship among the different faces of the protein; (x) loops in
which additional binding sites may be inserted are readily
identified through an examination of the three-dimensional
structure of a diabody (Zhu et al., 1996, High level secretion of a
humanized bispecific diabody from Escherichia coli, Biotechnology
(NY) 14(2): 192-96; Hudson et al., 1999, High avidity scFv
multimers; diabodies and triabodies, I. Immunol. Methods 231(1-2):
177-89). Taken together, these properties are advantageous for
using diabodies as structural elements for constructing complex,
multidimensional nanostructures.
[0161] scFv fragments can also associate into multivalent multimers
(Hudson et al., 1999, High avidity scFv multimers; diabodies and
triabodies, J. Immunol. Methods 231(1-2): 177-89; Power et al.,
2000, Synthesis of high avidity antibody fragments (scFv multimers)
for cancer imaging, J. Immunol. Methods 242(1-2): 193-204;
Todorovska et al., 2001, Design and application of diabodies,
triabodies and tetrabodies for cancer targeting, J. Immunol.
Methods 248(1-2): 47-66) (FIG. 3). Multimer formation is dependent
upon the length of the linker used to associate the variable
domains (V-domain) together, as well as the V-domain composition
and orientation (V.sub.H-V.sub.L versus V.sub.L-V.sub.H). Reducing
the linker length below three residues usually favors trimer or
triabody formation, e.g., scFv).sub.3. Tetrabody formation, e.g.,
(scFv).sub.4 also has been reported in at least two instances where
the linker length was 0 residues in length and the V-domain
orientation was V.sub.L-V.sub.H (Todorovska et al., 2001, Design
and application of diabodies, triabodies and tetrabodies for cancer
targeting, J. Immunol. Methods 248(1-2): 47-66).
[0162] An antibody variable domain may functionally comprise both a
structural element and a joining element in an assembly unit for
staged assembly. Like structural elements, the extent of a joining
element may not always be completely defined. For example, the
.beta.-sheet structure of an antibody variable domain maintains the
geometric relationship between the CDR and the other parts of the
molecule. But it is also important for maintaining the structural
relationships between the loops of the CDR that provide the binding
affinity and specificity of the complementary partner of the
joining pair. Consequently, an antibody variable domain may
functionally comprise both a structural element and a joining
element in an assembly unit. Thus, although antibody molecules and
binding fragments of antibodies are preferred elements of joining
elements, they may also provide structural framework for many
embodiments, and as described above, for an assembly unit.
[0163] Antibody Functional Elements
[0164] Antibody assembly units may include antibodies as functional
elements. Antibodies, binding fragments and binding derivatives as
described above that are not involved as a joining element or
structural element may serve as functional elements for site
specific attachment of other moieties. Thus, in certain
embodiments, an assembly unit having more than two joining elements
is used to build a nanostructure. The additional joining elements
can be used, for example: (i) as an attachment point for addition
or insertion of a functional element or functional moiety (see
Table 4 above); (ii) as the attachment point of the initiator to a
solid substrate; or (iii) as attachment points for
subassemblies.
[0165] Other Elements
[0166] In certain embodiments of the present invention, an assembly
unit comprises a structural element. Generally, the structural
element generally has a rigid structure (although in certain
embodiments, described below, the structural element may be
non-rigid). The structural element is preferably a defined peptide,
protein or protein fragment of known size and structure that
comprises at least about 50 amino acids and, generally, fewer than
2000 amino acids. Peptides, proteins and protein fragments are
preferred since naturally-occurring peptides, proteins and protein
fragments have well-defined structures, with structured cores that
provide stable spatial relationships between and among the
different faces of the protein. This property allows the structural
element to maintain pre-designed geometric relationships between
the joining elements and functional elements of the assembly unit,
and the relative positions and stoichiometries of assembly units to
which it is bound.
[0167] The use of proteins as structural elements has particular
advantages over other choices such as inorganic nanoparticles. Most
populations of inorganic nanoparticles are heterogeneous, making
them unattractive scaffolds for the assembly of a nanostructure. In
most populations, each inorganic nanoparticle is made up of a
different number of atoms, with different geometric relationships
between facets and crystal faces, as well as defects and
impurities. A comparably sized population of proteins is, by
contrast, very homogeneous, with each protein comprised of the same
number of amino acids, each arranged in approximately the same way,
differing in arrangement, for the most part, only through the
effect of thermal fluctuations. Consequently, two proteins designed
to interact with one another will always interact with the same
geometry, resulting in the formation of a complex of predictable
geometry and stoichiometry. This property is essential for
massively parallel "bottom-up" assembly of nanostructures.
[0168] A structural element may be used to maintain the geometric
relationships among the joining elements and functional elements of
a nanostructure. As such, a rigid structural element is generally
preferred for construction of nanostructures using the staged
assembly methods described herein. This rigidity is typical of many
proteins and may be conferred upon the protein through the
properties of the secondary structural elements making up the
protein, such as .alpha.-helices and .beta.-sheets.
[0169] Structural elements may be based on the structure of
proteins, protein fragments or peptides whose three-dimensional
structure is known or may be designed ab initio. Examples of
proteins or protein fragments that may be utilized as structural
elements in an assembly unit include, but are not limited to,
antibody domains, diabodies, single-chain antibody variable
domains, and bacterial pilins.
[0170] In some embodiments, structural elements, joining elements
and functional elements may be of well-defined extent, separated,
for example, by glycine linkers. In other embodiments, joining
elements may involve peptides or protein segments that are integral
parts of a structural element, or may comprise multiple loops at
one end of a structural element, such as in the case of the
complementarity determining regions (CDRs) of antibody variable
domains (Kabat et al., 1983, Sequences of Proteins of Immunological
Interest, U.S. Department of Health and Human Services). A CDR is a
joining element that is an integral part of the variable domain of
an antibody. The variable domain represents a structural element
and the boundary between the structural element and the CDR making
up the joining element (although well-defined in the literature on
the basis of the comparisons of many antibody sequences) may not
always be completely unambiguous structurally. There may not always
be a well-defined boundary between a structural element and a
joining element, and the boundary between these domains, although
well-defined on the basis of their respective utilities, may be
ambiguous spatially.
[0171] Structural elements of the present invention comprise, e.g.,
core structural elements of naturally-occurring proteins that are
then modified to incorporate joining elements, functional elements,
and/or a flexible domain (e.g., a tri-, tetra- or pentaglycine),
thereby providing useful assembly units. Consequently, in certain
embodiments, structures of existing proteins are analyzed to
identify those portions of the protein or part thereof that can be
modified without substantially affecting the rigid structure of
that protein or protein part.
[0172] For example, in certain embodiments, the amino acid sequence
of surface loop regions of a protein or structural element are
altered with little impact on the overall folding of the protein.
The amino acid sequences of a surface loop of a protein are
generally preferred as amino acid positions into which the
additional amino acid sequence of a joining element, a functional
element, and/or a flexible domain may be inserted, with the lowest
probability of disrupting the protein structure. Determining the
position of surface loops in a protein is carried out by
examination of the three-dimensional structure of the protein or a
homolog thereof, if three-dimensional atomic coordinates are
available, using, for example, a public-domain protein
visualization computer program such as RASMOL (Sayle et al., 1995,
RasMol: Biomolecular graphics for all, Trends Biochem. Sci. (TIBS)
20(9): 374-376; Saqi et al., 1994, PdbMotif--a tool for the
automatic identification and display of motifs in protein
structures, Comput. Appl. Biosci. 10(5): 545-46). In this manner,
amino acids included in surface loops, and the relative spatial
locations of these surface loops, can be determined.
[0173] If the three-dimensional structure of the protein being
engineered is not known, but that of a close homolog is known (as
is the case, for example, for essentially all antibody molecules),
the amino acid sequence of the molecule of interest, or a portion
thereof, can be aligned with that of the molecule whose
three-dimensional structure is known. This comparison (done, for
example, using BLAST (Altschul et al., 1997, Gapped BLAST and
PSI-BLAST: a new generation of protein database search programs,
Nucleic Acids Res. 25: 3389-3402) or LALIGN (Huang and Miller,
1991, A time efficient, linear-space local similarity algorithm,
Adv. Appl. Math. 12: 337-357) allows identification of all the
amino acids in the protein of interest that correspond to amino
acids that-constitute surface loops (.beta.-turns) in the protein
of known three-dimensional structure. In regions in which there is
high sequence similarity between the two proteins, this
identification is carried out with a high level of certainty. Once
a putative loop is identified and altered according to methods
disclosed herein, the resultant construct is tested to determine if
it has the expected properties. This analysis is performed even in
those instances where identification of the loop is highly
reliable, e.g. where that determination is based upon a known
three-dimensional protein structure.
[0174] Structural elements comprising leucine zipper-type coiled
coils in addition to antibody-derived domains can also be employed
in assembly units in the nanostructures of the invention. In
certain embodiments, the invention encompasses structural elements
comprising leucine zipper-type coiled coils for use in the
construction of nanostructures using the staged assembly methods of
the invention. Leucine zippers are well-known, a-helical protein
structures (Oas et al, 1994, Springs and hinges: dynamic coiled
coils and discontinuities, TIBS 19: 51-54; Branden et al., 1999,
Introduction to Protein Structure 2nd ed., Garland Publishing,
Inc., New York) that are involved in the oligomerization of
proteins or protein monomers into dimeric, trimeric, and tetrameric
structures, depending on the exact sequence of the leucine zipper
domain (Harbury et al., 1993, A switch between two-, three-, and
four-stranded coiled coils in GCN4 leucine zipper mutants, Science
262: 1401-07). While only dimers are disclosed herein for
simplicity, it would be apparent to one of ordinary skill in the
art that trimeric and tetrameric units may also be used for the
construction of assembly units for use in staged assembly of
nanostructures according to the methods disclosed herein. In
certain embodiments, trimeric and tetrameric units could be
especially useful for incorporation of functional elements that,
e.g., require two or more chemical moieties for proper activity,
for example, the incorporation of two cysteine moieties for binding
of gold particles.
[0175] Structural elements comprising four-helix bundles, for
example linked to antibody-derived domains, can also be employed in
assembly units in the nanostructures of the invention. The design
and construction of leucine zippers represent one type of a coiled
coil oligomerization peptide useful in the construction of a
structural element of an assembly unit. Another type is a
four-helix bundle, a non-limiting example of which is shown in FIG.
10. Because there are one or more loop segments (i.e. non-helical
segments) joining the helices to form an assembly unit, this
structure is also called a "helix-loop-helix" structure. The loop
sections contribute to the stability of the overall structure by
keeping the helices near each other and, therefore, at a
functionally high concentration. Examples of helix-loop-helix
proteins include, but are not limited to: the bacterial Rop protein
(a homodimer containing two helix-loop-helix molecules) (Lassalle
et al., 1998, Dimer-to-tetramer transformation: loop excision
dramatically alters structure and stability of the ROP four
alpha-helix bundle protein, J. Mol. Biol. 279(4): 987-1000); the
eukaryotic cytochrome b562 (a monomeric protein made up of a single
helix-loop-helix-loop-helix-loop-helix structure) (Lederer et al.,
1981, Improvement of the 2.5 .ANG. resolution model of cytochrome
b562 by redetermining the primary structure and using molecular
graphics, J. Mol. Biol. 148(4): 427-48); Max (Lavigne et al., 1998,
Insights into the mechanism of heterodimerization from the 1H-NMR
solution structure of the c-Myc-Max heterodimeric leucine zipper,
J. Mol. Biol. 281(1): 165-81); MyoD DNA-binding domain (Ma et al.,
1994, Crystal structure of MyoD bHLH domain-DNA complex:
perspectives on DNA recognition and implications for
transcriptional activation, Cell 77(3): 451-59); USF1 and USF2
DNA-binding domains (Ferre-D'Amare et al., 1994, Structure and
function of the b/HLH/Z domain of USF, EMBO J. 13(1): 180-9;
Kurschner et al., 1997, USF2/FIP associates with the b-Zip
transcription factor, c-Maf, via its bHLH domain and inhibits c-Maf
DNA binding activity, Biochem. Biophys. Res. Commun.231(2):
333-39); and Mit-f transcription factor DNA-binding domains (Rehli
et al., 1999, Cloning and characterization of the murine genes for
bHLH-ZIP transcription factors TFEC and TFEB reveal a common gene
organization for all MiT subfamily members, Genomics 56(1):
111-20).
[0176] Both helical regions and loop regions of the Rop protein
exhibit properties that indicate that the Rop protein, or fragments
thereof, may be used as structural elements in the construction of
assembly units in the staged assembly methods of the invention. In
one embodiment, the methods of Munson et al. (1996, What makes a
protein a protein? Hydrophobic core designs that specify stability
and structural properties, Protein Science 5: 1584-93) are used to
mutagenize the a and d residues in the helical regions of the Rop
protein to produce variant polypeptides having both increased and
decreased thermal stability.
[0177] In one aspect of the invention, functional elements include,
but are not limited to, peptides, proteins, protein domains, small
molecules, inorganic nanoparticles, atoms, clusters of atoms,
magnetic, photonic or electronic nanoparticles. The specific
activity or property associated with a particular functional
element, which will generally be independent of the structural
attributes of the assembly unit to which it is attached, can be
selected from a very large set of possible functions, including but
not limited to, a biological property such as those conferred by
proteins (e.g., a transcriptional, translational, binding,
modifying or catalyzing property). In other embodiments, functional
groups may be used that confer chemical, organic, physical
electrical, optical, structural, mechanical, computational,
magnetic or sensor properties to the assembly unit.
[0178] In another aspect of the invention, functional elements
include, but are not limited to: metallic or metal oxide
nanoparticles (Argonide Corporation, Sanford, Fla.; NanoEnergy
Corporation, Longmont, Colo.; Nanophase Technologies Corporation,
Romeoville, Ill.; Nanotechnologies, Austin, Tex.; TAL Materials,
Inc., Ann Arbor, Mich.); gold or gold-coated nanoparticles
(Nanoprobes, Inc., Yaphank, N.Y.; Nanospectra LLC, Houston Tex.);
immunoconjugates (Nanoprobes, Inc., Yaphank, N.Y.); non-metallic
nanoparticles (Nanotechnologies, Austin, Tex.); ceramic nanofibers
(Argonide Corporation, Sanford, Fla.); fullerenes or nanotubes
(e.g., carbon nanotubes) (Materials and Electrochemical Research
Corporation, Tucson, Ariz.; Nanolab, Brighton Mass.; Nanosys, Inc.,
Cambridge Mass.; Carbon Nanotechnologies Incorporated, Houston,
Tex.); nanocrystals (NanoGram Corporation, Fremont, Calif.; Quantum
Dot Corporation, Hayward Calif.); silicon or silicate nanocrystals
or powders (MTI Corporation, Richmond, Calif.); nanowires (Nanosys,
Inc., Cambridge Mass.); or quantum dots (Quantum Dot Corporation,
Hayward Calif.; Nanosys, Inc., Cambridge Mass.).
[0179] Functional elements may also comprise any art-known
detectable marker, including radioactive labels such as .sup.32P,
.sup.35S, .sup.3H, and the like; chromophores; fluorophores;
chemiluminescent molecules; or enzymatic markers.
[0180] In certain embodiment of this invention, a functional
element is a fluorophore. Exemplary fluorophore moieties that can
be selected as labels are set forth in Table 5.
5TABLE 5 Fluorophore Moieties That Can Be Used as Functional
Elements 4-acetamido-4'-isothiocyanatos- tilbene-2,2'disulfonic
acid acridine and derivatives: acridine acridine isothiocyanate
5-(2'-aminoethyl)aminonaph- thalene-1-sulfonic acid (EDANS)
4-amino-N-[3-vinylsulfonyl)phenyl]n- aphthalimide-3,5 disulfonate
(Lucifer Yellow VS)-(4-anilino-1-naphthyl)maleimide anthranilamide
Brilliant Yellow coumarin and derivatives: coumarin
7-amino-4-methylcoumarin (AMC, Coumarin 120)
7-amino-4-trifluoromethylcoumarin (Coumarin 151) Cy3 Cy5 cyanosine
4',6-diaminidino-2-phenylindole (DAPI)
5',5"-dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red)
7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin
diethylenetriamine pentaacetate 4,4'-diisothiocyanatodihydro-stilb-
ene-2,2'-disulfonic acid
4,4'-diisothiocyanatostilbene-2,2'-disulfo- nic acid
5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl
chloride) 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL)
4-dimethylaminophenylazophenyl-4'-isothiocyanate (DABITC) eosin and
derivatives: eosin eosin isothiocyanate erythrosin and derivatives:
erythrosin B erythrosin isothiocyanate ethidium fluorescein and
derivatives: 5-carboxyfluorescein (FAM)
5-(4,6-dichlorotriazin-2-yl)aminofluore- scein (DTAF)
2'7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE) fluorescein
fluorescein isothiocyanate QFITC (XRITC) fluorescamine IR144 IR1446
Malachite Green isothiocyanate 4-methylumbelliferone ortho
cresolphthalein nitrotyrosine pararosaniline Phenol Red
B-phycoerythrin o-phthaldialdehyde pyrene and derivatives: pyrene
pyrene butyrate succinimidyl 1-pyrene butyrate Reactive Red 4
(Cibacron .RTM. Brilliant Red 3B-A) rhodamine and derivatives:
6-carboxy-X-rhodamine (ROX) 6-carboxyrhodamine (R6G) lissamine
rhodamine B sulfonyl chloride rhodamine (Rhod) rhodamine B
rhodamine 110 rhodamine 123 rhodamine X isothiocyanate
sulforhodamine B sulforhodamine 101 sulfonyl chloride derivative of
sulforhodamine 101 (Texas Red)
N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA) tetramethyl
rhodamine tetramethyl rhodamine isothiocyanate (TRITC) riboflavin
rosolic acid terbium chelate derivatives
[0181] In other embodiments, a functional element is a
chemiluminescent substrate such as luminol (Amersham Biosciences),
BOLD.TM. APB (Intergen), Lumigen APS (Lumigen), etc.
[0182] In another embodiment, the functional element is an enzyme.
The enzyme, in certain embodiments, may produce a detectable signal
when a particular chemical reaction is conducted, such as the
enzymes alkaline phosphatase, horseradish peroxidase,
.beta.-galactosidase, etc.
[0183] In another embodiment, a functional element is a hapten or
an antigen (e.g., ras). In yet another embodiment, a functional
element is a molecule such as biotin, to which a labeled avidin
molecule or streptavidin may be bound, or digoxygenin, to which a
labeled anti-digoxygenin antibody may be bound.
[0184] In another embodiment, a functional element is a lectin such
as peanut lectin or soybean agglutinin. In yet another embodiment,
a functional element is a toxin, such as Pseudomonas exotoxin
(Chaudhary et al., 1989, A recombinant immunotoxin consisting of
two antibody variable domains fused to Pseudomonas exotoxin, Nature
339(6223): 394-97).
[0185] Peptides, proteins or protein domains may be added to
proteinaceous assembly units using the tools of molecular biology
commonly known in the art to produce fusion proteins in which the
functional elements are introduced at the N-terminus of the
proteins, the C-terminus of the protein, or in a loop within the
protein in such a way as to not disrupt folding of the protein.
Non-peptide functional elements may be added to an assembly unit by
the incorporation of a peptide or protein moiety that exhibits
specificity for said functional element, into the proteinaceous
portion of the assembly unit.
[0186] In another embodiment, an entire antibody variable domain
(e.g. a single-chain variable domain) is incorporated into an
assembly unit, e.g. into the joining or structural element thereof,
in order to act as an affinity target for a functional element. In
this embodiment, wherein an entire antibody variable domain is
inserted into a surface loop of, e.g., a joining element or a
structural element, a flexible segment (e.g., a polyglycine peptide
sequence) is preferably added to each side of the variable domain
sequence. This polyglycine linker will act as a flexible spacer
that facilitates folding of the original protein after synthesis of
the recombinant fusion protein. The antibody domain is chosen for
its binding specificity for a functional element, which can be, but
is not limited to, a protein or peptide, or to an inorganic
material.
[0187] In another embodiment of the present invention, a functional
element may be a quantum dot (semiconductor nanocrystal, e.g.,
QDOT.TM., Quantum Dot Corporation, Hayward, Calif.) with desirable
optical properties. A quantum dot can be incorporated into a
nanostructure through a peptide that has specificity for a
particular class of quantum dot. As would be apparent to one of
ordinary skill, identification of such a peptide, having a required
affinity for a particular type of quantum dot, is carried out using
methods well known in the art. For example, such a peptide is
selected from a large library of phage-displayed peptides using an
affinity purification method. Suitable purification methods
include, e.g., biopanning (Whaley et al., 2000, Selection of
peptides with semiconductor binding specificity for directed
nanocrystal assembly, Nature 405(6787): 665-68) and affinity column
chromatography. In each case, target quantum dots are immobilized
and the recombinant phage display library is incubated against the
immobilized quantum dots. Several rounds of biopanning are carried
out and phage exhibiting affinity for the quantum dots are
identified by standard methods after which the specificity of the
peptides are tested using standard ELISA methodology.
[0188] Typically, the affinity purification is an iterative process
that uses several affinity purification steps. Affinity
purification may been used to identify peptides with affinity for
particular metals and semiconductors (Belcher, 2001, Evolving
Biomolecular Control of Semiconductor and Magnetic Nanostructure,
presentation at Nanoscience: Underlying Physical Concepts and
Properties, National Academy of Sciences, Washington, D.C., May
18-20, 2001; Belcher et al., 2001, Abstracts of Papers, 222nd ACS
National Meeting, Chicago, Ill., United States, Aug. 26-30, 2001,
American Chemical Society, Washington, D.C.).
[0189] An alternate method is directed toward the use of libraries
of phage-displayed single chain variable domains, and to carry out
the same type of selection process. Accordingly, in certain
embodiments, a functional element is incorporated into a
nanostructure through the use of joining elements (interaction
sites) by which non-proteinaceous nanoparticles having desirable
properties are attached to the nanostructure. Such joining elements
are, in two non-limiting examples, derived from the complementarity
determining regions of antibody variable domains or from affinity
selected peptides.
[0190] Routine tests for electronic and photonic functional
elements that are commonly used to compare the electronic
properties of nanocrystals (single nanoparticles) and assemblies of
nanoparticles (Murray et al., 2000, Synthesis and characterization
of monodisperse nanocrystals and close-packed nanocrystal
assemblies, Ann. Rev. Material Science 30: 545-610), are used for
the analysis of nanostructures fabricated using the compositions
and methods disclosed herein.
[0191] In certain embodiments, the unique, tunable properties of
semiconductor nanocrystals make them preferable for use in
nanodevices, including photoconductive nanodevices and light
emitting diodes. The electrical properties of an individual
nanostructure are difficult to measure, and therefore,
photoconductivity is used as a measure of the properties of those
nanostructures. Photoconductivity is a well-known phenomena used
for analysis of the properties of semiconductors and organic
solids. Photoconductivity has long been used to transport electrons
between weakly interacting molecules in otherwise insulating
organic solids.
[0192] Photocurrent spectral responses may also be used to map the
absorption spectra of the nanocrystals in nanostructures and
compared to the photocurrent spectral responses of individual
nanocrystals (see, e.g., Murray et al., 2000, Synthesis and
characterization of monodisperse nanocrystals and close-packed
nanocrystal assemblies, Ann. Rev. Material Science 30: 545-610). In
addition, optical and photoluminescence spectra may also be used to
study the optical properties of nanostructures (see, e.g., Murray
et al., 2000, Synthesis and characterization of monodisperse
nanocrystals and close-packed nanocrystal assemblies, Ann. Rev.
Material Science 30: 545-610).
[0193] The greater the control exerted over the formation of arrays
of nanoparticles, the wider the array of optical, electrical and
magnetic phenomena that will be produced. With staged assembly of
nanostructures into which nanoparticles are incorporated with
three-dimensional precision, it is possible to control the
properties of solids formed therefrom in three dimensions, thereby
giving rise to a host of anisotropic properties useful in the
design of nanodevices. Routine tests and methods for characterizing
the properties of these assemblages are well-known in the art (see,
e.g., Murray et al., 2000, Synthesis and characterization of
monodisperse nanocrystals and close-packed nanocrystal assemblies,
Ann. Rev. Material Sci. 30: 545-610).
[0194] For example, biosensors are commercially available that are
made of a combination of proteins and quantum dots (Alivisatos et
al., 1996, Organization of `nanocrystal molecules` using DNA,
Nature 382: 609-11; Weiss et al., U.S. Pat. No. 6,207,392 entitled
"Semiconductor nanocrystal probes for biological applications and
process for making and using such probes," issued Mar. 27, 2001).
The ability to complex a quantum dot with a highly specific
biological molecule (e.g., a single stranded DNA or an antibody
molecule) provides a spectral fingerprint for the target of the
molecule. Using different sized quantum dots (each with very
different spectral properties), each complexed to a molecule with
different specificity, allows multiple sensing of components
simultaneously.
[0195] Inorganic structures such as quantum dots and nanocrystals
of metals or semiconductors may be used in the staged assembly of
nanostructures as termini of branches of the assembled
nanostructure. Once such inorganic structures are added, additional
groups cannot be attached to them because they have an
indeterminate stoichiometry for any set of binding sites engineered
into a nanostructure. This influences the sequence in which
assembly units are added to form a nanostructure being fabricated
by staged assembly. For example, once a particular nanocrystal is
added to the nanostructure, it is generally not preferred to add
additional assembly units with joining elements that recognize and
bind that type of nanocrystal, because it is generally not possible
to control the positioning of such assembly units relative to the
nanocrystal. Therefore, it may be necessary to add the nanocrystals
last, or at least after all the assembly units that will bind that
particular type of nanocrystal are added. In a preferred
embodiment, nanocrystals are added to nanostructures that are still
bound to a matrix and are sufficiently separated so that each
nanocrystal can only bind to a single nanostructure, thereby
preventing multiple cross-linking of nanostructures.
[0196] In one embodiment, a rigid nanostructure, fabricated
according to the staged assembly methods of the present invention,
comprises a magnetic nanoparticle attached as a functional element
to the end of a nanostructure lever arm, which acts as a very
sensitive sensor of local magnetic fields. The presence of a
magnetic field acts to change the position of the magnetic
nanoparticle, bending the nanostructure lever arm relative to the
solid substrate to which it is attached. The position of the lever
arm may be sensed, in certain embodiments, through a change in
position of, for example, optical nanoparticles attached as
functional elements to other positions (assembly units) along the
nanostructure lever arm. The degree of movement of the lever arm is
calibrated to provide a measure of the magnetic field.
[0197] In other embodiments, nanostructures that are fabricated
according to the staged assembly methods of the invention have
desirable properties in the absence of specialized functional
elements. In such embodiments, a staged assembly process provides a
two-dimensional or a three-dimensional nanostructure with small
(nanometer-scale), precisely-sized, and well-defined pores that can
be used, for example, for filtering particles in a microfluidic
system. In further aspects of this embodiment, nanostructures are
assembled that not only comprise such well-defined pores but also
comprise functional elements that enhance the separation properties
of the nanostructure, allowing separations based not only on size
but also with respect to the charge and/or hydrophilicity or
hydrophobicity properties of the molecules to be separated. Such
nanostructures can be used for HPLC separations, providing
extremely uniform packing materials and separations based upon
those materials. Examples of such functional elements include, but
are not limited to, peptide sequences comprising one or more side
chains that are positively or negatively charged at a pH used for
the desired chromatographic separation; and peptide sequences
comprising one or more amino acids having hydrophobic or lipophilic
side chains.
[0198] Junctions are architectural structures that can serve as
"switch points" in microelectronic circuits such as silicon based
electronic chips, etc. In certain embodiments, multivalent
antibodies or binding derivatives or binding fragments thereof are
used as junction structures and are introduced into nanostructures
using the methods of the present invention. One non-limiting
example of bioelectronic and biocomputational devices comprising
these nanostructure junctions are quantum cellular automata
(QCA).
[0199] Staged Assembly of Nanostructures
[0200] Antibody assembly units may be assembled to form
nanostructures by staged assembly. Staged assembly enables
massively parallel synthesis of complex, non-periodic,
multi-dimensional nanostructures in which organic and inorganic
moieties are placed, accurately and precisely, into a pre-designed,
three-dimensional architecture. In a staged assembly, a series of
assembly units is added in a given pre-designed order to an
initiator unit and/or nanostructure intermediate. Because a large
number of identical initiators are used and because subunits are
added to all initiators/intermediates simultaneously, staged
assembly fabricates multiple identical nanostructures in a
massively parallel manner. In preferred embodiments, the initiator
units are bound to a solid substrate, support or matrix. Additional
assembly units are added sequentially in a procedure akin to solid
phase polymer synthesis. The intermediate stage(s) of the
nanostructure while it is being assembled, and which comprises the
bound assembly units formed on the initiator unit, is generally
described as either a nanostructure intermediate or simply, a
nanostructure. Addition of each assembly unit to the nanostructure
intermediate undergoing assembly depends upon the nature of the
joining element presented by the previously added assembly unit and
is independent of subsequently added assembly units. Thus assembly
units can bind only to the joining elements exposed on the
nanostructure intermediate undergoing assembly; that is, the added
assembly units do not self-interact and/or polymerize.
[0201] Since the joining elements of a single assembly unit are
non-complementary and therefore do not interact with one another,
unbound assembly units do not form dimers or polymers. An assembly
unit to be added is preferably provided in molar excess over the
initiator unit or nanostructure intermediate in order to drive its
reaction with the intermediate to completion. Removal of unbound
assembly units during staged assembly is facilitated by carrying
out staged assembly using a solid-substrate-bound initiator so that
unbound assembly units can be washed away in each cycle of the
assembly process.
[0202] This scheme provides for assembly of complex nanostructures
using relatively few non-cross-reacting, complementary joining
pairs. Only a few joining pairs need to be used, since only a
limited number of joining elements will be exposed on the surface
of an assembly intermediate at any one step in the assembly
process. Assembly units with complementary joining elements can be
added and incubated against the nanostructure intermediate, causing
the added assembly units to be attached to the nanostructure
intermediate during an assembly cycle. Excess assembly units can
then be washed away to prevent them from forming unwanted
interactions with other assembly units during subsequent steps of
the assembly process. Each position in the nanostructure can be
uniquely defined through the process of staged assembly and
distinct functional elements can be added at any desired position.
The staged assembly method of the invention enables massive
parallel manufacture of complex nanostructures, and different
complex nanostructures can be further self-assembled into higher
order architectures in a hierarchic manner.
[0203] FIG. 11 depicts an embodiment of the staged assembly method
of the invention in one dimension. In step 1, an initiator unit is
immobilized on a solid substrate. In step 2, an assembly unit is
added to the initiator (i.e. the matrix bound initiator unit),
resulting in a nanostructure intermediate composed of two units.
Only a single assembly unit is added in this step, because the
second assembly unit cannot interact (i.e. polymerize) with
itself.
[0204] The initiator unit, or any of the assembly units
subsequently added during staged assembly including the capping
unit, may contain an added functional element and/or may comprise a
structural unit of different length from previously added units.
For example, in step 3 of FIG. 11, a third assembly unit is added
that comprises a functional element. In steps 4 and 5, additional
assembly units are added, each with a designed functional group.
Thus in the embodiment of staged assembly depicted in FIG. 11, the
third, fourth and fifth assembly units each carry a unique
functional element (designated by geometric shapes protruding from
the top of the assembly units in the figure).
[0205] The embodiment of staged assembly depicted in FIG. 11
requires only two non-cross-reacting, complementary joining pairs.
Self-assembly of the structure, as it stands at the end of step 5,
would require four non-cross-reacting, complementary joining pairs.
This relatively modest improvement in number of required joining
pairs becomes far greater as the size of the structure increases.
For instance, for a linear structure of N units assembled by an
extension of the five steps illustrated in FIG. 11, staged assembly
would still require only two non-cross-reacting, complementary
joining pairs, whereas self-assembly would require (N-1)
non-cross-reacting, complementary joining pairs.
[0206] The number of nanostructures fabricated is determined by the
number of initiator units bound to the matrix while the length of
each one-dimensional nanostructure is a function of the number of
assembly cycles performed. If assembly units with one or more
different functional elements are used, then the order of assembly
will define the relative spatial orientation of each functional
element relative to the other functional elements.
[0207] After each step in the method of staged assembly of the
invention, excess unbound assembly units are removed from the
attached nanostructure intermediate by a removal step, e.g., a
washing step. The substrate-bound nanostructure intermediate may be
washed with an appropriate solvent (e.g., an aqueous solution or
buffer). The solvent must be able to remove subunits held by
non-specific interactions without disrupting the specific,
interactions of complementary joining elements. Appropriate
solvents may vary as to pH, salt concentration, chemical
composition, etc., as required by the assembly units being
used.
[0208] A buffer used for washing the nanostructure intermediate can
be, for example, a buffer used in the wash steps implemented in
ELISA protocols, such as those described in Current Protocols in
Immunology (see Chapter 2, Antibody Detection and Preparation,
Section 2.1 "Enzyme-Linked Immunosorbent Assays," John Wiley &
Sons, 2001, Editors John E. Coligan, Ada M. Kruisbeek, David H.
Margulies, Ethan M. Shevach, Warren Strober, Series Editor: Richard
Coico).
[0209] In certain embodiments, an assembled nanostructure is
"capped" by addition of a "capping unit," which is an assembly unit
that carries only a single joining element. Furthermore, if the
initiator unit has been attached to the solid substrate via a
cleavable bond, the nanostructure can be removed from the solid
substrate and isolated. However, in some embodiments, the completed
nanodevice will be functional while attached to the solid substrate
and need not be removed.
[0210] The above-described steps of adding assembly units can be
repeated in an iterative manner until a complete nanostructure is
assembled, after which time the complete nanostructure can be
released by breaking the bond immobilizing the first assembly unit
from the matrix at a designed releasing moiety (e.g,. a protease
site) within the initiator unit or by using a pre-designed process
for release (e.g., lowering of pH). The process of staged assembly,
as illustrated in FIGS. 11 and 12 is one of the simplest
embodiments contemplated for staged assembly. In other embodiments,
assembly units with additional joining elements can be used to
create more complex assemblies. Assembly units may be added
individually or, in certain embodiments, they can be added as
subassemblies (FIG. 12). The result is a completely defined
nanostructure with functional elements that are distributed
spatially in relationship to one another to satisfy desired design
parameters. The compositions and methods disclosed herein provide
means for the assembly of these complex, designed nanostructures
and of more complex nanodevices formed by the staged assembly of
one or a plurality of nanostructures into a larger structure.
Fabrication of multidimensional nanostructures can be accomplished,
e.g., by incorporating precisely-spaced assembly units containing
additional joining elements into individual, one-dimensional
nanostructures, where those additional joining elements can be
recognized and bound by a suitable cross-linking agent to attach
the individual nanostructures together. In certain preferred
embodiments, such cross-linking could be, e.g., an antibody or a
binding derivative or a binding fragment thereof.
[0211] In some embodiments of the staged assembly method of the
invention, the initiator unit is tethered to a solid support. Such
tethering is not random (i.e., is not non-specific binding of
protein to plastic or random biotinylation of an assembly unit
followed by binding to immobilized streptavidin) but involves the
binding of a specific element of the initiator unit to the matrix
or substrate. The staged assembly process is a vectorial process
that requires an unobstructed joining element on the initiator unit
for attachment of the next assembly unit. Random binding of
initiator units to substrate would, in some cases, result in the
obstruction of the joining element needed for the attachment of the
next assembly unit, and thus lowering the number of initiator units
on which nanostructures are assembled.
[0212] In other embodiments of the staged assembly method of the
invention, the initiator unit is not immobilized to a solid
substrate. In this case, a removal step, e.g., a washing step, can
be carried out on a nanostructure constructed on a non-immobilized
or untethered initiator unit by: (1) attaching a magnetic
nanoparticle to the initiator unit and separating nanostructure
intermediates from non-bound assembly units by applying a magnetic
field; 2) separating the larger nanostructure intermediates from
unbound assembly units by centrifugation, precipitation or
filtration; or 3) in those instances in which a nanostructure
intermediate or assembled nanostructure is more resistant to a
destructive treatment (e.g., protease treatment or chemical
degradation), unbound assembly units are selectively destroyed.
[0213] Proteins have well-defined binding properties, and the
technology to manipulate the intermolecular interactions of
proteins is well known in the art (Hayashi et al., 1995, A single
expression system for the display, purification and conjugation of
single-chain antibodies, Gene 160(1): 129-30; Hayden et al., 1997,
Antibody engineering, Curr. Opin. Immunol. 9(2): 201-12; Jung et
al., 1999, Selection for improved protein stability by phage
display, J. Mol. Biol. 294(1): 163-80, Viti et al., 2000, Design
and use of phage display libraries for the selection of antibodies
and enzymes, Methods Enzymol. 326: 480-505; Winter et al., 1994,
Making antibodies by phage display technology, Annu. Rev. Immunol.
12: 433-55). The contemplated staged assembly of nanostructures,
however, need not be limited to components composed primarily of
biological molecules, e.g., proteins and nucleic acids, that have
specific recognition properties. The optical, magnetic or
electrical properties of inorganic atoms or molecules will be
required for some embodiments of nanostructures fabricated by
staged assembly.
[0214] There will be many embodiments of this invention in which
components not made up of proteins will be advantageously utilized.
In other embodiments, it may be possible to utilize the molecular
interaction properties of proteins or nucleic acids to construct
nanostructures composed of both organic and inorganic
materials.
[0215] In certain embodiments, inorganic nanoparticles are added to
components that are assembled into nanostructures using the staged
assembly methods of the invention. This may be done using joining
elements specifically selected for binding to inorganic particles.
For example, Whaley and co-workers have identified peptides that
bind specifically to semiconductor binding surfaces (Whaley et al.,
2000, Selection of peptides with semiconductor binding specificity
for directed nanocrystal assembly, Nature 405: 665-68). In one
embodiment, these peptides are inserted into protein components
described herein using standard cloning techniques. Staged assembly
of protein constructs as disclosed herein, provides a means of
distributing these binding sites in a rigid, well-defined
three-dimensional array.
[0216] Once the binding sites for a particular type of inorganic
nanoparticle are all in place, the inorganic nanoparticles can be
added using a cycle of staged assembly analogous to that used to
add proteinaceous assembly units. To accomplish this, it may be
necessary, in certain embodiments to adjust the solution conditions
under which the nanostructure intermediates are incubated, in order
to provide for the solubility of the inorganic nanoparticles. Once
an inorganic nanoparticle is added to the nanostructure
intermediate, it is not possible to add further units to the
inorganic nanoparticle in a controlled fashion because of the
microheterogeneities intrinsic to any population of inorganic
nanoparticles. These heterogeneities would render the geometry and
stoichiometry of further interactions uncontrollable.
[0217] FIG. 13 is a diagram illustrating the addition of protein
units and inorganic elements to a nanostructure according to the
staged assembly methods of the invention. In step 1, an initiator
unit is bound to a solid substrate. In step 2, an assembly unit is
bound specifically to the initiator unit. In step 3, an additional
assembly unit is bound to the nanostructure undergoing assembly.
This assembly unit comprises an engineered binding site specific
for a particular inorganic element. In step 4, the inorganic
element (depicted as a cross-hatched oval) is added to the
structure and bound by the engineered binding site. Step 5 adds
another assembly unit with a binding site engineered for
specificity to a second type of inorganic element, and that second
inorganic element (depicted as a hatched diamond) is added in step
6.
[0218] The order in which assembly units are added is determined by
the desired structure and/or activity that the product
nanostructure, and the need to minimize the number of
cross-reacting joining element pairs used in the assembly process.
Hence determining the order of assembly is an integral part of the
design of a nanostructure to be fabricated by staged assembly.
Joining elements are chosen, by design, to permit staged assembly
of the desired nanostructure. Since the choice of joining
element(s) is generally independent of the functional elements to
be incorporated into the nanostructure, the joining elements are
mixed and matched as needed to fabricate assembly units with the
necessary functional elements and joining elements that will
provide for the placement of those functional elements in the
desired spatial orientation.
[0219] For example, assembly units comprising two joining elements,
designed using the six joining elements that make up three joining
pairs, can include any of 18 pairs of the joining elements that are
non-interacting. There are 21 possible pairs of joining elements,
but three of these pairs are interacting (e.g. A-A') and their use
in an assembly unit would lead to the self-association of identical
assembly units with one another. In the example illustrated below,
joining elements are denoted as A, A', B, B', C and C', where A and
A', B and B', and C and C' are complementary pairs of joining
elements joining pairs), i.e. they bind to each other with
specificity, but not to any of the other four joining elements
depicted. Six representative assembly units, each of which
comprises two joining elements, wherein each joining element
comprises a non-identical, non-complementary joining element, are
depicted below. In this depiction, each assembly unit further
comprises a unique functional element, one of a set of six, and
represented as F.sub.1 to F.sub.6. According to these conventions,
six possible assembly units can be designated as:
[0220] A-F.sub.1-B
[0221] B'-F.sub.2-A'
[0222] B'-F.sub.3-C'
[0223] C-F.sub.4-B
[0224] B'-F.sub.5-A'
[0225] A-F.sub.6-C'
[0226] Staged assembly according to the methods disclosed herein
can be used to assemble the following illustrative linear,
one-dimensional nanostructures, in which the order and relative
vectorial orientation of each assembly unit is independent of the
order of the functional elements (the symbol .circle-solid.--is
used to represent the solid substrate to which the initiator is
attached and a double colon represents the specific interaction
between assembly units):
[0227]
.circle-solid.-A-F1-B::B'-F2-A'::A-F1-B::B'-F2-A'::A-F1-B::B'-F2-A'-
::A-F1-B::B'-F2-A'
[0228]
.circle-solid.-A-F1-B::B'-F2-A'::A-F6-C'::C-F4-B::B'-F2-A'::A-F1-B:-
:B'-F5-A'::A-F6-C'
[0229]
.circle-solid.-A-F1-B::B'-F2-A'::A-F1-B::B'-F5-A'::A-F1-B::B'-F2-A'-
::A-F1-B::B'-F3-C'
[0230]
.circle-solid.-A-F1-B::B'-F3-C'::C-F4-B::B'-F3-C'::C-F4-B::B'-F3-C'-
::C-F4-B::B'-F2-A'
[0231] As is apparent from this illustration, a large number of
unique assembly units can be constructed using a small number of
complementary joining elements. Moreover, only a small number of
complementary joining elements are required for the fabrication of
a large number of unique and complex nanostructures, since only one
type of assembly unit is added in each staged assembly cycle and,
therefore, joining elements can be used repeatedly without
rendering ambiguous the position of an assembly unit within the
completed nanostructure.
[0232] In each of the cases illustrated above, only two or three
joining pairs have been used. Self-assembly of any of these
structures would require the use of seven non-cross-reacting
joining pairs. If these linear structures were N units in extent,
they would still only require two or three joining pairs, but for
self-assembly, they would require (N-1) non-cross-reacting,
complementary joining pairs.
[0233] In another aspect of the invention, by interchanging the
positions of the two joining elements of an assembly unit depicted
above, the spatial position and orientation of the attached
functional element will be altered within the overall structure of
the nanostructure fabricated. This aspect of the invention
illustrates yet another aspect of the design flexibility provided
by staged assembly of nanostructures as disclosed herein.
[0234] Attachment of each assembly unit to an initiator or
nanostructure intermediate is mediated by formation of a specific
joining-pair interaction between one joining element of the
assembly unit and one or more unbound complementary joining
elements carried by the initiator or nanostructure intermediate. In
many embodiments, only a single unbound complementary joining
element will be present on the initiator or nanostructure
intermediate. However, in other embodiments, it may be advantageous
to add multiple identical assembly units to multiple sites on the
assembly intermediate that comprise identical joining elements. In
these embodiments, the staged assembly proceeds by the parallel
addition of assembly units, but only a single unit will be attached
at any one site on the intermediate, and assembly at all sites that
are involved will occur in a pre-designed, vectorial manner.
[0235] Structural integrity of the nanostructure is of critical
importance throughout the process of staged assembly, and the
assembly units are preferably connected by non-covalent
interactions. A specific non-covalent interaction is, for example,
an interaction that occurs between an assembly unit and a
nanostructure intermediate. The specific interaction should exhibit
adequate affinity to confer stability to the complex between the
assembly unit and the nanostructure intermediate sufficient to
maintain the interaction stably throughout the entire staged
assembly process. A specific non-covalent interaction should
exhibit adequate specificity such that the added assembly unit will
form stable interactions only with joining elements designed to
interact with it. The interactions that occur among elements during
the staged assembly process disclosed herein are preferably
operationally "irreversible." A binding constant that meets this
requirement cannot be defined unambiguously since "irreversible" is
a kinetic concept, and a binding constant is based on equilibrium
properties. Nevertheless, interactions with Kd's of the order of
10.sup.-7 or lower (i.e. higher affinity and similar to the Kd of a
typical diabody-epitope complex) will typically act "irreversibly"
on the time scale of interest, i.e. during staged assembly of a
nanostructure.
[0236] The intermolecular interactions need not act "irreversibly,"
however, on the timescale of the utilization of a nanostructure
(i.e. its shelf life or working life expectancy). In certain
embodiments, nanostructures fabricated according to the staged
assembly methods disclosed herein are subsequently stabilized by
chemical fixation (e.g., by fixation with paraformaldehyde or
glutaraldehyde) or by cross-linking. The most common schemes for
cross-linking two proteins involve the indirect coupling of an
amine group on one assembly unit to a thiol group on a second
assembly unit (see, e.g., Handbook of Fluorescent Probes and
Research Products, Eighth Edition, Chapter 2, Molecular Probes,
Inc., Eugene, Oreg.; Loster et al., 1997, Analysis of protein
aggregates by combination of cross-linking reactions and
chromatographic separations, J. Chromatogr. B. Biomed. Sci. Appl.
699(1-2): 439-61; Phizicky et al., 1995, Protein-protein
interactions: methods for detection and analysis, Microbiol. Rev.
59(1): 94-123).
[0237] In certain embodiments of the invention, the fabrication of
a nanostructure by the staged assembly methods of the present
invention involves joining relatively rigid and stable assembly
units, using non-covalent interactions between and among assembly
units. Nevertheless, the joining elements that are incorporated
into useful assembly units can be rather disordered, that is,
neither stable nor rigid, prior to interaction with a second
joining element to form a stable, preferably rigid, joining pair.
Therefore, in certain embodiments of the invention, individual
assembly units may include unstable, flexible domains prior to
assembly, which, after assembly, will be more rigid. In preferred
embodiments, a nanostructure fabricated using the compositions and
methods disclosed herein is a rigid structure.
[0238] According to the methods of the invention, analysis of the
rigidity of a nanostructure, as well as the identification of any
architectural flaws or defects, are carried out using methods
well-known in the art, such as electron microscopy.
[0239] In another embodiment, structural rigidity can be tested by
attaching one end of a completed nanostructure directly to a solid
surface, i.e., without the use of a flexible tether. The other end
of the nanostructure (or a terminal branch of the nanostructure, if
it is a multi-branched structure) is then attached to an atomic
force microscope (AFM) tip, which is movable. Force is applied to
the tip in an attempt to move it. If the nanostructure is flexible,
there will be an approximately proportional relationship between
the force applied and tip movement as allowed by deflection of the
nanostructure. In contrast, if the nanostructure is rigid, there
will be little or no deflection of the nanostructure and tip
movement as the level of applied force increases, up until the
point at which the rigid nanostructure breaks. At that point, there
will be a large movement of the AFM tip even though no further
force is applied. As long as the attachment points of the two ends
are stronger than the nanostructure, this method will provide a
useful measurement of rigidity.
[0240] According to the present invention, each position in a
nanostructure is distinguishable from all others, since each
assembly unit can be designed to interact tightly, specifically,
and uniquely with its neighbors. Each assembly unit can have an
activity and/or characteristic that is distinct to its position
within the nanostructure. Each position in the nanostructure is
uniquely defined through the process of staged assembly, and
through the properties of each assembly unit and/or functional
element that is added at a desired position. In addition, the
staged-assembly methods and assembly units disclosed herein are
amenable to large scale, massively parallel, automated
manufacturing processes for construction of complex nanostructures
of well-defined size, shape, and function.
[0241] The methods and compositions of the present invention
capitalize upon the precise dimensions, uniformity and diversity of
spatial geometries that proteins are capable of that are used in
the construction of the assembly units employed herein.
Furthermore, as described hereinbelow, the methods of the invention
are advantageous because genetic engineering techniques can be used
to modify and tailor the properties of those biological materials
used in the methods of the invention disclosed herein, as well as
to synthesize large quantities of such materials in
microorganisms.
[0242] Initiator Assembly Units
[0243] An initiator assembly unit is the first assembly unit
incorporated into a nanostructure that is formed by the staged
assembly method of the invention. An initiator assembly unit may be
attached, in certain embodiments, by covalent or non-covalent
interactions, to a solid substrate or other matrix. An initiator
assembly unit is also known as an "initiator unit."
[0244] Staged assembly of a nanostructure begins by the
non-covalent, vectorial addition of a selected assembly unit to the
initiator unit. According to the methods of the invention, an
assembly unit is added to the initiator unit through (i) the
incubation of an initiator unit, which in some embodiments, is
immobilized to a matrix or substrate, in a solution comprising the
next assembly unit to be added. This incubation step is followed by
(ii) a removal step, e.g., a washing step, in which excess assembly
units are removed from the proximity of the initiator unit.
[0245] Assembly units bind to the initiator unit through the
formation of specific, non-covalent bonds. The joining elements of
the next assembly unit are chosen so that they attach only at
pre-designated sites on the initiator unit. Only one assembly unit
can be added to a target joining element on the initiator unit
during the first staged-assembly cycle, and binding of the assembly
unit to the target initiator unit is vectorial. Staged assembly
continues by repeating steps (i) and (ii) until all of the desired
assembly units are incorporated into the nanostructure according to
the desired design of the nanostructure.
[0246] In a preferred embodiment of the staged assembly method of
the invention, an initiator unit is immobilized on a substrate and
additional units are added sequentially in a procedure analogous to
solid phase polymer synthesis.
[0247] An initiator unit is a category of assembly unit, and
therefore can comprise any of the structural, joining, and/or
functional elements described hereinbelow as being comprised in an
assembly unit of the invention. An initiator unit can therefore
comprise any of the following molecules, or a binding derivative or
binding fragment thereof: a monoclonal antibody; a multispecific
antibody, a Fab or F(ab').sub.2 fragment, a single-chain antibody
fragment (scFv); a bispecific, chimeric or bispecific heterodimeric
F(ab').sub.2; a diabody or multimeric scFv fragment; a bacterial
pilin protein, a leucine zipper-type coiled coil, a four-helix
bundle, a peptide epitope, or a PNA, or any other type of assembly
unit disclosed herein.
[0248] In certain embodiments, the invention provides an initiator
assembly unit which comprises at least one joining element. In
other embodiments, the invention provides an initiator assembly
unit with two or more joining elements.
[0249] Initiator units may be tethered to a matrix in a variety of
ways. The choice of tethering method will be determined by several
design factors including, but not limited to: the type of initiator
unit, whether the finished nanostructure must be removed from the
matrix, the chemistry of the finished nanostructure, etc. Potential
tethering methods include, but are not limited to, antibody binding
to initiator epitopes, His tagged initiators, initiator units
containing matrix binding domains (e.g., chitin-binding domain,
cellulose-binding domain), antibody binding proteins (e.g., protein
A or protein G) for antibody or antibody-derived initiator units,
streptavidin binding of biotinylated initiators, PNA tethers, and
specific covalent attachment of initiators to matrix.
[0250] In certain embodiments, an initiator unit is immobilized on
a solid substrate. Initiator units may be immobilized on solid
substrates using methods commonly used in the art for
immobilization of antibodies or antigens. There are numerous
methods well known in the art for immobilization of antibodies or
antigens. These methods include non-specific adsorption onto
plastic ELISA plates; biotinylation of a protein, followed by
immobilization by binding onto streptavidin or avidin that has been
previously adsorbed to a plastic substrate (see, e.g., Sparks et
al., 1996, Screening phage-displayed random peptide libraries, in
Phage Display of Peptides and Proteins, A Laboratory manual,
editors, B. K. Kay, J. Winter and J. McCafferty, Academic Press,
San Diego, pp. 227-53). In addition to ELISA microtiter plates,
protein may be immobilized onto any number of other solid supports
such as Sepharose (Dedman et al., 1993, Selection of target
biological modifiers from a bacteriophage library of random
peptides: the identification of novel calmodulin regulatory
peptides, J. Biol. Chem. 268; 23025-30) or paramagnetic beads
(Sparks et al., 1996, Screening phage-displayed random peptide
libraries, in Phage Display of Peptides and Proteins, A Laboratory
manual, editors, B. K. Kay, J. Winter and J. McCafferty, Academic
Press, San Diego, pp. 227-53). Additional methods that may be used
include immobilization by reductive amination of amine-containing
biological molecules onto aldehyde-containing solid supports
(Hermanson, 1996, Bioconjugate Techniques, Academic Press, San
Diego, p. 186), and the use of dimethyl pimelimidate (DMP), a
homobifunctional cross-linking agent that has imidoester groups on
either end (Hermanson, 1996, Bioconjugate Techniques, Academic
Press, San Diego, pp. 205-06). This reagent has found use in the
immobilization of antibody molecules to insoluble supports
containing bound protein A (e.g., Schneider et al., 1982, A
one-step purification of membrane proteins using a high efficiency
immunomatrix, J. Biol. Chem. 257, 10766-69).
[0251] In a specific embodiment, an initiator unit is a diabody
that comprises a tethering domain (T) that recognizes and binds an
immobilized antigen/hapten and an opposing domain (A) to which
additional assembly units are sequentially added in a staged
assembly. Antibody 8F5, which is directed against the antigenic
peptide VKAETRLNPDLQPTE (SEQ ID NO: 1) derived human rhinovirus
(Serotype 2) viral capsid protein Vp2, is used as the T domain
(Tormo et al., 1994, Crystal structure of a human rhinovirus
neutralizing antibody complexed with a peptide derived from viral
capsid protein VP2, EMBO J. 13(10): 2247-56). The A domain is the
same lysozyme anti-idiotopic antibody (E5.2) previously described
for Diabody Unit 1. The completed initiator assembly unit therefore
contains 8F5.times.730.1.4 (T.times.A ) as the opposing CDRs. The
initiator unit is constructed and functionally characterized using
the methods described herein for characterizing joining elements
and/or structural elements comprising diabodies.
[0252] In order to immobilize the initiator unit onto a solid
support matrix, the rhinovirus antigenic peptide may fused to the
protease recognition peptide factor Xa through a short flexible
linker spliced at the N termini of the Factor Xa sequence, IEGR,
(Nagai and Thogersen, 1984, Generation of beta-globin by
sequence-specific proteolysis of a hybrid protein produced in
Escherichia coli, Nature309(5971): 810-12) and between the Factor
Xa sequence and the antigenic peptide sequence. This fusion peptide
may be covalently linked to CH-Sepharose 4B (Pharmacia); a
sepharose derivative that has a six-carbon long spacer arm and
permits coupling via primary amines. (Alternatively, Sepharose
derivatives for covalent attachment via carboxyl groups may be
used.) The covalently attached fusion protein will serve as a
recognition epitope for the tethering domain "8F5" in the initiator
unit (T.times.A).
[0253] Once the initiator is immobilized, additional diabody units
(diabody assembly units 1 and 2) may be sequentially added in a
staged assembly, unidirectionally from binding domain A'. Upon
completion of the staged assembly, the nanostructure may be either
cross-linked to the support matrix or released from the matrix upon
addition of the protease Factor Xa. The protease will cleave the
covalently attached antigenic/Factor Xa fusion peptide, releasing
the intact nanostructure from the support matrix, since, by design,
there are no Factor Xa recognition sites contained within any of
the designed protein assembly units.
[0254] An alternate strategy of cleaving the peptide fusion from
the solid support matrix that does not require the addition of
Factor Xa, can also be implemented. This method utilizes a
cleavable spacer arm attached to the sepharose matrix. The antigen
peptide is covalently attached through a phenyl-ester linkage to
the matrix. Once the immobilized antibody binds initiator assembly
unit, the initiator assembly unit remains tethered to the support
matrix until chemical cleavage of the spacer arm with
imidazoleglycine buffer at pH 7.4 at which point the initiator
unit/antigen complex (and associated nanostructure) are released
from the support matrix.
[0255] Methods for Characterizing Joining Elements
[0256] Methods for Identifying Joining--Element Interactions by
Antibody-Phage-Display Technology
[0257] In certain embodiments of the invention, joining elements
suitable for use in the methods of the invention are screened and
their interactions identified using antibody-phage-display
technology. Phage-display technology for production of recombinant
antibodies, or binding derivatives or binding fragments thereof,
can be used to produce proteins capable of binding to a broad range
of diverse antigens, both organic and inorganic (e.g. proteins,
peptides, nucleic acids, sugars, and semiconducting surfaces,
etc.). Methods for phage-display technology are well known in the
art (see, e.g., Marks et al., 1991, By-passing immunization: human
antibodies from V-gene libraries displayed on phage, J. Mol. Biol.
222: 581-97; Nissim et al., 1994, Antibody fragments from a "single
pot" phage display library as immunochemical reagents, EMBO J. 13:
692-98; De Wildt et al., 1996, Characterization of human variable
domain antibody fragments against the U1 RNA-associated A protein,
selected from a synthetic and patient derived combinatorial V gene
library, Eur. J. Immunol. 26: 629-39; De Wildt et al., 1997, A new
method for analysis and production of monoclonal antibody fragments
originating from single human B-cells, J. Immunol. Methods. 207:
61-67; Willems et al., 1998, Specific detection of myeloma plasma
cells using anti-idiotypic single chain antibody fragments selected
from a phage display library, Leukemia 12: 1295-1302; van Kuppevelt
et al., 1998, Generation and application of type-specific
anti-heparin sulfate antibodies using phage display technology,
further evidence for heparin sulfate heterogeneity in the kidney,
J. Biol. Chem. 273: 12960-66; Hoet et al., 1998, Human monoclonal
autoantibody fragments from combinatorial antibody libraries
directed to the U1snRNP associated U1C protein, epitope mapping,
immunolocalization and V-gene usage, Mol. Immunol. 35:1045-55).
[0258] Whereas recombinant antibody technology permits the
isolation of antibodies with known specificity from hybridoma
cells, it does not allow for the rapid creation of specific mAbs.
Separate immunizations, followed by cell fusions to generate
hybridomas are required to generate each mAb of interest. This can
be time consuming as well as laborious.
[0259] In preferred embodiments, antibody-phage-display technology
is used to overcome these limitations, so that mAbs that recognize
particular antigens of interest can be generated more effectively
(for methods, see Winter et al., 1994, Making antibodies by phage
display technology, Ann. Rev. Immunol. 12: 433-55; Hayashi et al.,
1995, A single expression system for the display, purification and
conjugation of single-chain antibodies, Gene 160(1): 129-30;
McGuinness et al., 1996, Phage diabody repertoires for selection of
large numbers of bispecific antibody fragments, Nat. Biotechnol.
14(9): 1149-54; Jung et al., 1999, Selection for improved protein
stability by phage display, J. Mol. Biol. 294(1): 163-80;Viti et
al., 2000, Design and use of phage display libraries for the
selection of antibodies and enzymes, Methods Enzymol. 326:
480-505). Generally, in antibody-phage-display technology, the Fv
or Fab antigen-binding portions of V.sub.L and the V.sub.H genes
are "rescued" by PCR amplification using the appropriate primers,
from cDNA derived from human spleen or human peripheral blood
lymphocyte cells. The rescued V.sub.L and the V.sub.H gene
repertoires (DNA sequences) are spliced together and inserted into
the minor coat protein of a bacteriophage (e.g., M13 or fd, or a
binding derivative thereof) to create a fusion bacteriophage coat
protein (Chang et al., 1991, Expression of antibody Fab domains on
bacteriophage surfaces. Potential use for antibody selection, J.
Immunol. 147(10): 3610-14; Kipriyanov and Little, 1999, Generation
of recombinant antibodies, Mol. Biotechnol. 12(2): 173-201). The
resulting bacteriophage contain a functional antibody fused to the
outer surface of the phage protein coat and a copy of the gene
fragment encoding the antibody V.sub.L and V.sub.H incorporated
into the phage genome.
[0260] Using these methods, bacteriophage displaying antibodies
that have affinity towards a particular antigen of interest can be
isolated by, e.g., affinity chromatography, via the binding of a
population of recombinant bacteriophage carrying the displayed
antibody to a target epitope or antigen, which is immobilized on a
solid surface or matrix. Repeated cycles of binding, removal of
unbound or weakly-bound phage particles, and phage replication
yield an enriched population of bacteriophage carrying the desired
V.sub.L and V.sub.H gene fragments.
[0261] Antigens of interest may include peptides, proteins,
immunoglobulin constant regions, CDRs (for production of
anti-idiotypic antibodies) other macromolecules, haptens, small
molecules, inorganic particles and surfaces.
[0262] Once purified, the linked V.sub.L and V.sub.H gene fragments
can be rescued from the bacteriophage genome by standard DNA
molecular techniques known in the art, cloned and expressed. The
number of antibodies created by this method is directly correlated
to the size and diversity of the gene repertoire and offers an
optimal method by which to create diverse antibody libraries that
can be screened for antigenicity towards virtually any target
molecule. mAbs that have been created by antibody-phage-display
technology often demonstrate specific binding towards antigen in
the picomolar to nanomolar range (Sheets et al., 1998, Efficient
construction of a large nonimmune phage antibody library: the
production of high-affinity human single-chain antibodies to
protein antigens, Proc. Natl. Acad. Sci. USA 95(11): 6157-62).
[0263] Antibodies, or binding derivatives or binding fragments
thereof, that are useful in the methods of the invention may be
selected using an antibody or fragment phage display library
constructed and characterized as described above. Such an approach
has the advantage of providing methods for efficiently screening a
library having a high complexity (e.g. 10.sup.9), so as to
dramatically increase identification of antibodies or fragments
suitable for use in the methods of the invention.
[0264] In certain embodiments, methods for cloning an
immunoglobulin repertoire ("repertoire cloning") are used to
produce an antibody for use in the staged-assembly methods of the
invention. Repertoire cloning may be used for the production of
virtually any kind of antibody without involving an
antibody-producing animal. Methods for cloning an immunoglobulin
repertoire ("repertoire cloning") are well known in the art, and
can be performed entirely in vitro. In general, to perform
repertoire cloning, messenger RNA (mRNA) is extracted from B
lymphocytes obtained from peripheral blood. The mRNA serves as a
template for cDNA synthesis using reverse transcriptase and
standard protocols (see, e.g., Clinical Gene Analysis and
Manipulation, Tools, Techniques and Troubleshooting, Sections IA,
IC, IIA, IB, IIC and IIIA, Editors Janusz A. Z. Jankowski, Julia M.
Polak, Cambridge University Press 2001; Sambrook et al., 2001,
Molecular Cloning, A Laboratory Manual, Third Edition, Chapters 7,
11, 14 and 18, Cold Spring Harbor Laboratory Press, N.Y.; Ausubel
et al., 1989, Current Protocols in Molecular Biology, Chapters 3,
4, 11, 15 and 24, Green Publishing Associates and Wiley
Interscience, NY). Immunoglobulin cDNAs are specifically amplified
by PCR, using the appropriate primers, from this complex mixture of
cDNA. In order to construct immunoglobulin fragments with the
desired binding properties, PCR products from genes encoding
antibody light (L) and heavy (H) chains are obtained. The products
are then introduced into a phagemid vector. Cloned genes or gene
fragments incorporated into the bacteriophage genome as fusions
with a phage coat protein, are expressed in a suitable bacterial
host leading to the synthesis of a hybrid scFv immunoglobulin
molecule that is carried on the surface of the bacteriophage.
Therefore the bacteriophage population represents a mixture of
immunoglobulins with all specificities included in the
repertoire.
[0265] Antigen-specific immunoglobulin is selected from this
population by an iterative process of antigen immunoadsorption
followed by phage multiplication. A bacteriophage specific only for
an antigen of interest will remain following multiple rounds of
selection, and may be introduced into a new vector and/or host for
further engineering or to express the phage-encoded protein in
soluble form and in large amounts.
[0266] Antibody phage display libraries can thus be used, as
described above, for the isolation, refinement, and improvement of
epitope-binding regions of antibodies that can be used as joining
elements in the construction of assembly units for use in the
staged assembly of nanostructures, as disclosed herein.
[0267] Methods for Characterizing Joining--Element Interactions
Using X-ray Crystallography
[0268] In many instances, molecular recognition between proteins or
between proteins and peptides may be determined experimentally. In
one aspect of the invention, the protein-protein interactions that
define the joining element interactions, and are critical for
formation of a joining pair are characterized and identified by
X-ray crystallographic methods commonly known in the art. Such
characterization enables the skilled artisan to recognize joining
pair interactions that may be useful in the compositions and
methods of the present invention.
[0269] Methods for Characterizing Joining--Element Specificity and
Affinity
[0270] Verification that two complementary joining elements
interact with specificity may be established using, for example,
ELISA assays, analytical ultracentrifugation, or BIAcore
methodologies (Abraham et al., 1996, Determination of binding
constants of diabodies directed against prostate-specific antigen
using electrochemiluminescence-based immunoassays, J. Mol.
Recognit. 9(5-6): 456-61; Atwell et al., 1996, Design and
expression of a stable bispecific scFv dimer with affinity for both
glycophorin and N9 neuraminidase, Mol. Immunol. 33(17-18): 1301-12;
Muller et al. 1998), A dimeric bispecific mini-antibody combines
two specificities with avidity, FEBS Lett. 432(1-2): 45-49), or
other analogous methods well known in the art, that are suitable
for demonstrating and/or quantitating the strength of
intermolecular binding interactions.
[0271] Design and Engineering of Structural, Joining and Functional
Elements
[0272] Design of structural, joining and functional elements of the
invention, and of the assembly units that comprise them, is
facilitated by analysis and determination of those structures in
the desired binding interaction, as revealed in a defined crystal
structure, or through homology modeling based on a known crystal
structure of a highly homologous material. Design of a useful
assembly unit comprising one or more functional elements preferably
involves a series of decisions and analyses that may include, but
are not limited to, some or all of the following steps:
[0273] (i) selection of the functional elements to be incorporated
based on the desired overall function of the nanostructure;
[0274] (ii) selection of the desired geometry based on the target
function, in particular, determination of the relative positions of
the functional elements;
[0275] (iii) selection of joining elements through determination,
identification or selection of those peptides or proteins, e.g.
from a combinatorial library, that have specificity for the
functional nanoparticles to be incorporated into the desired
nanostructure;
[0276] (iv) based on the needed separations between functional
elements comprising, e.g. nanoparticles such as quantum dots, etc.,
selection of structural elements that will provide a suitably rigid
structure with correct dimensions and having positions for
incorporation of joining elements with the correct geometry and
stoichiometry;
[0277] (v) design of fusion proteins incorporating peptide or
protein joining elements, from step (iii) and the structural
element selected in step (iv) such that the folding of the
structural and joining elements of the assembly unit are not
disrupted (e.g., through incorporation at .beta.-turns);
[0278] (vi) computer modeling of the resultant fusion proteins in
the context of the overall design of the nanostructure and refining
of the design to optimize the structural dimensions as required by
the functional specifications; or
[0279] (vii) design of the assembly sequence for staged
assembly.
[0280] Modification of a structural element protein, for example,
usually involves insertion, deletion, or modification of the amino
acid sequence of the protein in question. In many instances,
modifications involve insertions or substitutions to add joining
elements not extant in the native protein. A non-limiting example
of a routine test to determine the success of an insertion mutation
is a circular dichroism (CD) spectrum. The CD spectrum of the
resultant fusion mutant protein can be compared to the CD of the
native protein.
[0281] If the insert is small (e.g., a short peptide), then the
spectra of a properly folded insertion mutant will be very similar
to the spectra of the native protein. If the insertion is an entire
protein domain (e.g. single chain variable domain), then the CD
spectrum of the fusion protein should correspond to the sum of the
CD spectra of the individual components (i.e. that of the native
protein and fusion protein comprising the native protein and the
functional element). This correspondence provides a routine test
for the correct folding of the two components of the fusion
protein.
[0282] Preferably, a further test of the successful engineering of
a fusion protein is made. For example, an analysis may be made of
the ability of the fusion protein to bind to all of its targets,
and therefore, to interact successfully with all joining pairs.
This may be performed using a number of appropriate ELISA assays;
at least one ELISA is performed to test the affinity and
specificity of the modified protein for each of the joining pairs
required to form the nanostructure.
[0283] Uses of the Staged-Assembly Method and of Nanostructures
Constructed Thereby
[0284] The staged-assembly methods and the assembly units of the
invention have use in the construction of myriad nanostructures.
The uses of such nanostructures are readily apparent and include
applications that require highly regular, well-defined arrays of
one-, two-, and three-dimensional structures such as fibers, cages,
or solids, which may include specific attachment sites that allow
them to associate with other materials.
[0285] In certain embodiments, the nanostructures fabricated by the
staged assembly methods of the invention are one-dimensional
structures. For example, nanostructures fabricated by staged
assembly can be used for structural reinforcement of other
materials, e.g., aerogels, paper, plastics, cement, etc. In certain
embodiments, nanostructures that are fabricated by staged assembly
to take the form of long, one-dimensional fibers are incorporated,
for example, into paper, cement or plastic during manufacture to
provide added wet and dry tensile strength.
[0286] In another embodiment, the nanostructure is a patterned or
marked fiber that can be used for identification or recognition
purposes. In such embodiments, the nanostructure may contain such
functional elements as e.g., a fluorescent dye, a quantum dot, or
an enzyme.
[0287] In a further embodiment, a particular nanostructure is
impregnated into paper and fabric as an anti-counterfeiting marker.
In this case, a simple color-linked antibody reaction (such as
those commercially available in kits) is used to verify the origin
of the material. Alternatively, such a nanostructure could bind
dyes, inks or other substances, either before or after
incorporation, to color the paper or fabrics or to modify their
appearance or properties in other ways.
[0288] In another embodiment, nanostructures are incorporated, for
example, into ink or dyes during manufacture to increase solubility
or miscibility.
[0289] In another embodiment, a one-dimensional nanostructure e.g.,
a fiber, bears one or more enzyme or catalyst functional elements
in desired positions. The nanostructure serves as a support
structure or scaffold for an enzymatic or catalytic reaction to
increase its efficiency. In such an embodiment, the nanostructure
may be used to "mount" or position enzymes or other catalysts in a
desired reaction order to provide a reaction "assembly line."
[0290] In another embodiment, a one-dimensional nanostructure,
e.g., a fiber, is used as an assembly jig. Two or more components,
e.g., functional units, are bound to the nanostructure, thereby
providing spatial orientation. The components are joined or fused,
and then the resultant fused product is released from the
nanostructure.
[0291] In another embodiment, a nanostructure is a one-, two- or
three-dimensional structure that is used as a support or framework
for mounting nanoparticles (e.g., metallic or other particles with
thermal, electronic or magnetic properties) with defined spacing,
and is used to construct a nanowire or nanocircuit.
[0292] In another embodiment, the staged assembly methods of the
invention are used to accomplish electrode-less plating of a
one-dimensional nanostructure (fiber) with metal to construct a
nanowire with a defined size and/or shape. For example, a
nanostructure could be constructed that comprises metallic
particles as functional elements.
[0293] In another embodiment, a one-dimensional nanostructure
(e.g., a fiber) comprising magnetic particles as functional
elements is aligned by an external magnetic field to control fluid
flow past the nanostructure. In another embodiment, the external
magnetic field is used to align or dealign a nanostructure (e.g.,
fiber) comprising optical moieties as functional elements for use
in LCD-type displays.
[0294] In another embodiment, a nanostructure is used as a size
standard or marker of precise dimensions for electron
microscopy.
[0295] In other embodiments, the nanostructures fabricated by the
staged assembly methods of the invention are two- or
three-dimensional structures. For example, in one embodiment, the
nanostructure is a mesh with defined pore size and can serve as a
two-dimensional sieve or filter.
[0296] In another embodiment, the nanostructure is a
three-dimensional hexagonal array of assembly units that is
employed as a molecular sieve or filter, providing regular vertical
pores of precise diameter for selective separation of particles by
size. Such filters can be used for sterilization of solutions
(i.e., to remove microorganisms or viruses), or as a series of
molecular-weight cut-off filters. In this embodiment, the protein
components of the pores, such as structural elements or functional
elements, may be modified so as to provide specific surface
properties (i.e., hydrophilicity or hydrophobicity, ability to bind
specific ligands, etc.). Among the advantages of this type of
filtration device is the uniformity and linearity of pores and the
high pore to matrix ratio.
[0297] It will be apparent to one skilled in the art that the
methods and assembly units disclosed herein may be used to
construct a variety of two- and three-dimensional structures such
as polygonal structures (e.g., octagons), as well as open solids
such as tetrahedrons, icosahedrons formed from triangles, and boxes
or cubes formed from squares and rectangles (e.g., the cube
disclosed in Section 11, Example 6). The range of structures is
limited only by the types of joining and functional elements that
can be engineered on the different axes of the structural
elements.
[0298] In another embodiment, a two-or three-dimensional
nanostructure may be used to construct a surface coating comprising
optical, electric, magnetic, catalytic, or enzymatic moieties as
functional units. Such a coating could be used, for example, as an
optical coating. Such an optical coating could be used to alter the
absorptive or reflective properties of the material coated.
[0299] A surface coating constructed using nanostructures of the
invention could also be used as an electrical coating, e.g., as a
static shielding or a self-dusting surfaces for a lens (if the
coating were optically clear). It could also be used as a magnetic
coating, such as the coating on the surface of a computer hard
drive.
[0300] Such a surface coating could also be used as a catalytic or
enzymatic coating, for example, as surface protection. In a
specific embodiment, the coating is an antioxidant coating.
[0301] In another embodiment, the nanostructure may be used to
construct an open framework or scaffold with optical, electric,
magnetic, catalytic, enzymatic moieties as functional elements.
Such a scaffold may be used as a support for optical, electric,
magnetic, catalytic, or enzymatic moieties as described above. In
certain embodiments, such a scaffold could comprise functional
elements that are arrayed to form thicker or denser coatings of
molecules, or to support soluble micron-sized particles with
desired optical, electric, magnetic, catalytic, or enzymatic
properties.
[0302] In another embodiments, a nanostructure serves as a
framework or scaffold upon which enzymatic or antibody binding
domains could be linked to provide high density multivalent
processing sites to link to and solubilize otherwise insoluble
enzymes, or to entrap, protect and deliver a variety of molecular
species.
[0303] In another embodiment, the nanostructure may be used to
construct a high density computer memory with addressable
locations.
[0304] In another embodiment, the nanostructure may be used to
construct an artificial zeolite, i.e., a natural mineral (hydrous
silicate) that has the capacity to absorb ions from water, wherein
the design of the nanostructure promotes high efficiency processing
with reactant flow-through an open framework.
[0305] In another embodiment, the nanostructure may be used to
construct an open framework or scaffold that serves as the basis
for a new material, e.g., the framework may possess a unique
congruency of properties such as strength, density, determinate
particle packing and/or stability in various environments.
[0306] In certain embodiments, the staged-assembly methods of the
invention can also be used for constructing computational
architectures, such as quantum cellular automata (QCA) that are
composed of spatially organized arrays of quantum dots. In QCA
technology, the logic states are encoded by positions of individual
electrons, contained in QCA cells composed of spatially positioned
quantum dots, rather than by voltage levels. Staged assembly can be
implemented in an order that spatially organizes quantum dot
particles in accordance with the geometries necessary for the
storage of binary information. Examples of logic devices that can
be fabricated using staged assembly for the spatially positioning
and construction of QCA cells for quantum dot cellular automata
include QCA wires, QCA inverters, majority gates and full adders
(Amlani et al., 1999, Digital logic gate using quantum-dot cellular
automata, Science 284(5412): 289-91; Cowburn and Welland, 2000),
Room temperature magnetic quantum cellular automata, Science
287(5457): 1466-68; Orlov et al., 1997, Realization of a Functional
Cell for Quantum-Dot Automata, Science 277: 928-32).
[0307] The invention will now be further described with reference
to the following, non-limiting examples.
EXAMPLE 1
[0308] Staged-Assembly of a Nanostructure Having a Joining Element
Comprising a Peptide Epitope
[0309] This example discloses staged assembly using monovalent Fab
fragments ("Fab1" and "Fab2,") each with a different peptide
epitope fused at their C-terminus (FIG. 4).
[0310] The CDR of Fab1 has specificity for the peptide fused to the
C-terminus of Fab2. Likewise, the CDR of Fab2 has specificity for
the peptide fused to the C-terminus of Fab1.
[0311] The two joining pairs provide specific interactions between
these two assembly units. The first Fab can be immobilized to a
solid substrate using standard methods. This surface can then be
incubated with a solution containing Fab2 which has fused a peptide
exhibiting specificity for Fab1. This incubation will result in the
formation of a nanostructure intermediate comprised of one copy of
Fab1 (immobilized) and one copy of Fab2. The intermediate can then
be incubated against a solution containing Fab1, resulting in the
formation of an intermediate comprised of a copy of Fab1 attached
to a copy of Fab2 that is sequentially attached to a copy of Fab1.
This assembly process may then continue iteratively for as long as
is necessary to achieve the size of linear structure required.
[0312] Assembly unit-1 is a monovalent assembly unit comprising an
antibody Fab fragment with CDR (CDR1) that specifically binds to
peptide 2 with a linked C-terminal peptide epitope (peptide 1).
[0313] Assembly unit-2 is a monovalent assembly unit comprising an
antibody Fab fragment with CDR (CDR2) that specifically binds to
peptide 1 with a linked C-terminal peptide epitope (peptide 2).
[0314] Joining Pairs.
6 Joining pair 1: Joining element peptide 1 interacts with joining
element CDR 2. Joining pair 2: Joining element peptide 2 interacts
with joining element CDR 1.
[0315]
7 Staged Assembly Steps Procedure Step 1 a) Add assembly unit-1 b)
Wash Step 2 a) Add assembly unit-2 b) Wash Step 3 a) Repeat Step 1
Step 4 a) Repeat Step 2
EXAMPLE 2
[0316] Staged Assembly using Multispecific Protein Assembly
Units
[0317] This example discloses an embodiment of the staged assembly
methods of the invention that uses multispecific protein assembly
units. Permutations and combinations of multispecific protein
assembly units may be used for the construction of complex one-,
two-, and three-dimensional macromolecular nanostructures,
including, for example, the staged assembly illustrated in FIG. 14,
which utilizes bivalent and tetravalent assembly units.
[0318] Staged assembly of a nanostructure comprising a four-point
junction only requires a minimum of five assembly units and four
joining pairs. The five assembly units required include four
bispecific and one tetraspecific assembly unit. In this example,
the joining pairs employed to join adjacent assembly units are
idiotope/anti-idiotope in nature. A minimum of four such
idiotope/anti-idiotope joining pairs are needed for staged-assembly
in this example.
[0319] (a) Assembly Units
[0320] In FIG. 14:
[0321] Assembly unit-1 is a bivalent protein assembly unit
comprising a non-interacting (idiotope/anti-idiotope) joining pair
A and B.
[0322] Assembly unit-2 is a bivalent assembly unit comprising a
non-interacting (idiotope/anti-idiotope) joining pair B' and
A'.
[0323] Assembly unit-3 is a tetravalent assembly unit comprising
non-interacting (idiotope/anti-idiotope) joining pair B' and A' and
non-interacting (idiotope/anti-idiotope) joining pair C and D.
[0324] Assembly unit-4 is a bivalent assembly unit comprising a
non-interacting (idiotope/anti-idiotope) joining pair C' and A.
[0325] Assembly unit-5 is a bivalent assembly unit with
non-interacting (idiotope/anti-idiotope) joining pair D' and
B'.
[0326] (b) Complementary Joining Pairs
[0327] A interacts with A' in complementary joining pair 1.
[0328] B interacts with B' in complementary joining pair 2.
[0329] C interacts with C' in complementary joining pair 3.
[0330] D interacts with D' in complementary joining pair 4.
[0331] (c) Protocol for Staged Assembly using Multispecific Protein
Assembly Units
[0332] The following steps of staged assembly are illustrated in
FIG. 14. The resultant nanostructure is illustrated FIG. 14, Step
11.
8 Staged Assembly Steps Procedure Step 1 a) Add assembly unit-1 b)
Wash Step 2 a) Add assembly unit-2 b) Wash Step 3 a) Repeat Step 1
Step 4 a) Add assembly unit-3 b) Wash Step 5 a) Repeat Step 1 Step
6 a) Add assembly unit-4 b) Wash Step 7 a) Repeat Step 2 Step 8 a)
Add assembly unit-5 b) Wash Step 9 a) Repeat Step 1 Step 10 a)
Repeat Step 2 Step 11 a) Repeat Step 1
EXAMPLE 3
[0333] Fabrication of a Macromolecular Nanostructure
[0334] To build a macromolecular assembly, two assembled
nanostructures intermediates can be joined to one another using the
staged assembly methods of the invention. This example describes
the fabrication of a macromolecular nanostructure from two
nanostructure intermediates.
[0335] FIG. 15 illustrates the staged assembly of the two
nanostructure intermediates fabricated from the staged assembly
protocol illustrated in FIG. 14. Nanostructure intermediate-1 is
illustrated as Step-11 tin FIG. 14. Nanostructure intermediate-2 is
illustrated as Step-8 in FIG. 14. The protocol in Section 9.1 below
describes the addition of two nanostructure intermediates by the
association of a complementary joining pair.
[0336] Protocol for the Addition of Two Nanostructure Intermediates
by the Association of a Complementary Joining Pair
[0337] The following steps of staged assembly are illustrated in
FIG. 15. The resultant macromolecular nanostructure is illustrated
FIG. 15, Step 5.
9 Staged Assembly Steps Procedure Step 1 Steps 1-11 of staged
assembly protocol described above in Section 8 (Example 3) Step 2
a) Add A' capping unit b) Wash Step 3 Remove nanostructure
intermediate-1 from the support matrix and isolate Step 4 Perform
Steps 1-8 of staged assembly protocol described above in Section 8
(Example 3), leaving nanostructure intermediate-2 attached to the
support matrix Step 5 a) Add nano structure intermediate-1 b)
Wash
EXAMPLE 4
[0338] Demonstration of Self-Assembly and Staged Assembly of a
Bivalent and Bispecific Diabody Joining Pair
[0339] Demonstration of Self-Assembly
[0340] As disclosed hereinabove, staged assembly may be carried out
using two non-cross-reacting diabody assembly unit constructs that
are expressed and purified. Solutions of each diabody unit protein
alone should remain clear, since the single diabody assembly units
will not self-polymerize (i.e., self-assemble).
[0341] If the two solutions are mixed, however, the diabody units
are capable of oligomerization as linked units and form long fibers
in which the two diabody units alternate (FIG. 1). This
self-assembly is readily observable by eye, by simple light
scattering or turbidity experiments and can be readily confirmed by
electron microscopy of negatively stained polymer rods.
[0342] Demonstration of Staged Assembly
[0343] Staged assembly is carried out by immobilizing the initiator
to a sepharose solid support matrix and then contacting the
matrix-bound initiator with diabody assembly unit-1. This is
followed by a wash step, in which excess diabody unit-1 is removed
from the bound nanostructure (containing the initiator unit and
bound diabody unit-1). The nanostructure is then incubated with
diabody assembly unit-2, followed by washing and incubating in the
presence of additional copies of diabody assembly unit-1, etc.,
through a number of cycles (FIG. 11). Electron microscopy is used
to determine the length and geometry of the polymers assembled
through different numbers of binding and wash cycles. These lengths
are precisely proportional to the number of cycles.
EXAMPLE 5
[0344] Analysis of Polymerization by Light Scattering
[0345] The extent polymerization of macromolecular monomers, such
as the diabodies used in this example, may be analyzed by light
scattering. Light scattering measurements from a light scattering
photometer, e.g., the DAWN-DSP photometer (Wyatt Technology Corp.,
Santa Barbara, Calif.), provides information for determination of
the weight average molecular weight, determination of particle
size, shape and particle-particle pair correlations.
EXAMPLE 6
[0346] Molecular Weight Determination (Degree of Polymerization) by
Sucrose Gradient Sedimentation
[0347] Linked diabody units of different lengths sediment at
different rates in a sucrose gradient in zonal ultracentrifugation.
The quantitative relationship between the degree of polymerization
and sedimentation in Svedberg units is then calculated. This method
is useful for characterizing the efficiency of self-assembly in
general, as well as the process of staged assembly at each step of
addition of a new diabody unit.
EXAMPLE 7
[0348] Morphology and Length of Rods by Electron Microscopy
[0349] After sucrose gradient fractionation and SDS-PAGE analysis,
the partially purified fractions containing rods are apparent.
Samples of the appropriate fractions are placed on EM grids and
stained or shadowed to look for large structures using electron
microscopy in order to determine their morphology.
EXAMPLE 8
[0350] Staged Assembly of a Three-Dimensional Cube
[0351] This example discloses the fabrication of a
three-dimensional cubic structure by staged assembly from assembly
units comprising structural elements from engineered triabody and
diabody fragments. The joining elements of the assembly units are
the multispecific binding domains of triabodies or diabodies.
[0352] Triabodies are trivalent and make up the vertices of the
cubic-like structure. Diabodies are bivalent and, in this example,
two are used to construct the edges of the cubic structure, thereby
spanning the space between the triabodies.
[0353] In the case of the initiator unit, an added peptide epitope
is engineered as a joining element within the triabody structural
element for immobilization to a solid support (and defined as the
first vertex of the cube in the staged assembly). Therefore the
joining elements for the triabody initiator unit comprise four
non-complementary joining elements, three of which are comprised of
the trispecific binding domains of the triabody and the fourth from
a peptide epitope engineered within the triabody structure designed
specifically to interact with solid support matrix. The peptide
epitope comprised in the initiator unit can be engineered to
contain a pre-designed releasing moiety (e.g. a protease site) that
can be cleaved from the initiator unit and joined to the
nanostructure from the solid support matrix upon complete
nanofabrication of the three-dimensional nanocube. Since the
three-dimensional structure of a triabody has been well
characterized (Pei et al., 1997, The 2.0-.ANG. resolution crystal
structure of a trimeric antibody fragment with noncognate
V.sub.H-V.sub.L domain pairs shows a rearrangement of V.sub.HCDR3,
Proc. Natl. Acad. Sci. USA 94(18): 9637-42), the insertion points
within the protein structure can be identified for engineering
additional joining elements, as discussed hereinabove, by visual
investigation of the available X-ray coordinates.
[0354] Another triabody comprised of three trispecific binding
domains as the joining elements makes up another assembly unit (the
other 7 vertices of the cube). The other assembly units, namely the
diabody units comprised of two bispecific binding domains as
joining elements, will form the edges of the cube (edges can be
defined as the vectorial lattices between defined vertices of the
cube). Each edge of the cube will be fabricated from two diabody
assembly units). In this example, a total of 32 assembly units are
required for the nanofabrication of a three-dimensional nanocube: 8
triabodies (one initiator unit and 7 assembly units making up the 8
vertices) and 24 diabodies (all assembly units making up the 12
edges). A total of 7 non-cross-reacting, complementary joining
pairs required for the fabrication of the nanocube.
[0355] Triabodies are three dimensional, equilateral triangle
prism-shaped proteins that contain one joining element (CDR) at
each of the three vertices. Diabodies, on the other hand, are
rectangular prism shaped proteins with two opposing joining
elements (CDRs). The nanofabrication of a three-dimensional (3-D)
cube composed of triabodies and diabodies requires geometric and
spatial relationships of the associated assembly units to be within
defined design specifications of the three-dimensional cube shown
in FIG. 16.
[0356] Particular geometries and spatial orientations of associated
triabodies and Fab fragments have been physically characterized
(Lawrence et al., 1998, Orientation of antigen binding sites in
dimeric and trimeric single chain Fv antibody fragments, FEBS Lett.
425(3): 479-84). The three Fab arms, when associated to the
vertices of a triabody, are not coplanar but, instead, are angled
together in one direction and appear as the legs of a tripod
(Lawrence et al., 1998, Orientation of antigen binding sites in
dimeric and trimeric single chain Fv antibody fragments, FEBS Lett.
425(3): 479-84). The angles between adjacent Fab arms associated to
the triabody was measured to be between 80-136.degree. (i.e this
falls within the required geometric and spatial relationships of
the associated assembly units for the formation of a vertex
associated with three edges of a cube) and that of a diabody and a
Fab fragment associations was measured between 60 and 180.degree.
(this falls within the required geometric and spatial relationships
for the formation of one edge of the cube upon the association
(joining) of two adjacent diabody elements). The angle between
planar edges of the cube is defined as 90.degree. and that of a
cubic edge as 180.degree.. Therefore, utilizing triabodies as the
vertices of a cube and diabodies as the edges, taking into
consideration the limited structural flexibility inherent within
antibody fragments, and the characteristic geometrical and spatial
associations of antibody fragments observed, it will be possible to
construct a three-dimensional cube as disclosed herein.
[0357] The cube is constructed by first identifying 7
non-cross-reacting, complementary joining element pairs. In this
embodiment, idiotope/anti-idiotope pairs are constructed using
standard methods disclosed above. The 14 joining elements that are
elements of these pairs are incorporated into bispecific diabodies
and trispecific triabodies as indicated by the architecture
disclosed below. FIG. 16 is a diagram of the assembly of a cubic
structure with the joining pairs indicated by letters (A being
complementary to A'; B complementary to B', etc.); and the order of
assembly indicated by numbers. The first unit is the initiator
unit, and it is indicated by the number `1`, and comprises joining
elements A, B and C. The second unit (`2`) comprises joining
elements A' and D. When a surface on which a unit 1 is immobilized
is incubated with a solution containing element 2, the element will
be added to the complementary binding site `A` on unit 1 resulting
in a nanostructure intermediate comprising units 1 and 2. After
washing off excess copies of assembly unit 2, the intermediate is
incubated against assembly unit 3, comprising joining elements D'
and A. This unit will bind with specificity to the complementary
joining element on unit 2, resulting in a nanostructure
intermediate comprising units 1, 2, and 3. This process is then
continued with alternating steps of incubation and washing, until
the entire structure is formed. Since 32 assembly units are added
one at a time, there will be 31 steps in the assembly process (not
counting the immobilization of unit 1 to a solid substrate).
[0358] A key element in planning a staged assembly of a
nanostructure is the tracking of which joining elements are exposed
after each step in the process. In the assembly of this nanocubic
structure, the following joining elements are exposed after each
step:
10 Last added unit Joining elements exposed 1 A B C 2 D B C 3 A B C
4 A D C 5 A B C 6 A B D 7 A B C 8 E F B C 9 E D B C 10 E F B C 11 E
F B A G 12 E F B D G 13 E F B A G 14 C E B G 15 C D B G 16 C E B G
17 C E A F G 18 C E D F G 19 C E A F G 20 C B F G 21 C D F G 22 C B
F G 23 D B F G 24 C B F G 25 A F G 26 A F D 27 A F G 28 A D G 29 A
F G 30 D F G 31 A F G 32 -- -- --
[0359] After unit 32 is added, no joining elements are exposed.
[0360] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the invention in addition to those described
herein will become apparent to those skilled in the art from the
foregoing description. Such modifications are intended to fall
within the scope of the appended claims.
[0361] All references cited herein are incorporated herein by
reference in their entirety and for all purposes to the same extent
as if each individual publication, patent or patent application was
specifically and individually indicated to be incorporated by
reference in its entirety for all purposes.
[0362] The citation of any publication is for its disclosure prior
to the filing date and should not be constructed as an admission
that the present invention is not entitled to antedate such
publication by virtue of prior invention.
* * * * *